Tumour-specific animal proteins

ABSTRACT

CASB7439 polypeptides and polynucleotides, immunogenic compositions comprising them and methods for producing such polypeptides by recombinant techniques are disclosed. Also disclosed are methods for utilizing CASB7439 polypeptides and polynucleotides in diagnostics, and vaccines for prophylactic and therapeutic treatment of cancers, particularly colorectal cancers, autoimmune diseases, and related conditions.

CROSS REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/301,895 filed Dec. 13, 2005, now issued as U.S. Pat. No. 7,803,379,which is a continuation of U.S. patent application Ser. No. 10/226,872filed Aug. 23, 2002, which was filed as a continuation-in-part ofInternational Application No. PCT/EP01/01779, filed on Feb. 16, 2001,which claims priority of Great Britain Patent Application No. 0004269.7,filed Feb. 23, 2000, which claims priority of Great Britain PatentApplication No. 0009905.1; filed Apr. 20, 2000, which claims priority ofGreat Britain Patent Application No. 0021080.7, filed Aug. 25, 2000.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

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THE NAMES OF THE PARTIES TO A JOINT RESEARCH AGREEMENT

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INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ON A COMPACT DISC

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BACKGROUND OF THE INVENTION

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BRIEF SUMMARY OF THE INVENTION

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BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

FIG. 1: shows Real-time PCR data using the Taqman probe. The legend isas follows: Adrenal gland: Ad_Gl; Bladder: Bl; Bone marrow: Bo_Ma;Cervix: Ce; Colon: Co; Fallopian tube: Fa_Tu; Ileon: Il; Liver: Li;Lung: Lu; Lymph node: Ly_No; Oesophagus: Oe; Parathyroid gland: Pa_Thy;Placenta: Pl; Prostate: Pr; Rectum: Re; Skin: Sk; Skeletal muscle:Sk_Mu; Small intestine: Sm_In; Spleen: Sp; Testis: Te; Thyroid gland:Thy; Trachea: Tr.

FIG. 2 shows Real-time PCR expression using Sybr protocol. The legend isas follows: Adrenal gland: Ad_GI; Bladder: Bl; Bone marrow: Bo_Ma;Cervix: Ce; Colon: Co; Lymph node: Ly_No; Oesophagus: Oe; Parathyroidgland: Pa_Thy; Placenta: Pl; Prostate: Pr; Rectum: Re; Skin: Sk;Skeletal muscle: Sk_Mu; Small intestine: Sm_In; Spleen: Sp; Testis: Te;Thyroid gland: Thy; Trachea: Tr; Heart: He.

FIG. 3 shows Coomassie blue stained SDS PAGE of the cell extract fromthe strain expressing CASB7439. Lane 1 shows the molecular markers, lane2 the cellular extract induced 5 h at 39° C.; lane 3 shows thesupernatant of cellular extract induced; and lane 4 shows the pellet ofcellular extract induced.

FIG. 4 shows a Western blot analysis of NS1-CASB7439 expressed protein.The gel is loaded with the cell extract from the strain expressingCASB7439 and revealed with anti-NS1 monoclonal antibody.

FIG. 5 shows a Coomassie-blue stained SDS-PAGE of CASB7439 afterpurification. Lanes 1 and 5 represent the molecular weight markers;lanes 2, 3, 4 are respectively loaded with 2 μl, 4 μl and 6 μl ofpurified protein.

FIG. 6 shows a Western blot CASB7439 after purification as revealed byan anti-polyhistidine monoclonal antibody.

FIG. 7 shows IHC results on colon tumour #9476 biospy.

FIG. 8 shows IHC results colon normal mucosa #9476 biospy.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to pharmaceutical compositions and methodsfor inducing an immune response against tumours-related antigens. Morespecifically, the invention relates to polynucleotides, herein referredto as CASB7439 polynucleotides, polypeptides encoded thereby (referredto herein as CASB7439 polypeptides), recombinant materials and methodsfor their production. In another aspect, the invention relates tomethods for using such polypeptides and polynucleotides, including thetreatment of cancer, more particularly colorectal cancer, and autoimmunediseases and other related conditions. In another aspect, the inventionrelates to pharmaceutical compositions containing CASB7439 polypeptidesand polynucleotides, to methods of manufacture of such compositions andto their use in medicine. In a further aspect, the invention relates tomethods for identifying agonists and antagonists/inhibitors using thematerials provided by the invention, and treating conditions associatedwith CASB7439 polypeptide imbalance with the identified compounds. In astill further aspect, the invention relates to diagnostic assays fordetecting diseases associated with inappropriate CASB7439 polypeptideactivity or levels.

Polypeptides and polynucleotides of the present invention are believedto be important immunogens for specific prophylactic or therapeuticimmunization against tumours, because they are specifically expressed orhighly over-expressed in tumours compared to normal cells and can thusbe targeted by antigen-specific immune mechanisms leading to thedestruction of the tumour cell. They can also be used to diagnose theoccurrence of tumour cells. Furthermore, their inappropriate expressionin certain circumstances can cause an induction of autoimmune,inappropriate immune responses, which could be corrected throughappropriate vaccination using the same polypeptides or polynucleotides.In this respect the most important biological activities to our purposeare the antigenic and immunogenic activities of the polypeptide of thepresent invention. A polypeptide of the present invention may alsoexhibit at least one other biological activity of a CASB7439polypeptide, which could qualify it as a target for therapeutic orprophylactic intervention different from that linked to the immuneresponse.

In a first aspect, the present invention relates to CASB7439polypeptides. Such peptides include isolated polypeptides, comprising anamino acid sequence which has at least 70% identity, preferably at least80% identity, more preferably at least 90% identity, yet more preferablyat least 95% identity, most preferably at least 97-99% identity, to thatof SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:11,SEQ ID NO:12 or SEQ ID NO:14 over the entire length of SEQ ID NO:2, SEQID NO:3, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 or SEQ IDNO:14 respectively, with the proviso that said isolated polypeptide isnot SEQ ID NO:2, SEQ ID NO:12 or SEQ ID NO:14. Such polypeptides includethose comprising the amino acid of SEQ ID NO:3, SEQ ID NO:7, SEQ IDNO:10 and SEQ ID NO:11.

Further peptides of the present invention include isolated polypeptides,in which the amino acid sequence has at least 70% identity, preferablyat least 80% identity, more preferably at least 90% identity, yet morepreferably at least 95% identity, most preferably at least 97-99%identity, to the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:7, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 or SEQ ID NO:14 over theentire length of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:10,SEQ ID NO:11, SEQ ID NO:12 or SEQ ID NO:14 respectively, with theproviso that said isolated polypeptide is not SEQ ID NO:2, SEQ ID NO:12or SEQ ID NO:14. Such polypeptides include the polypeptides of SEQ IDNO:3, SEQ ID NO:7, SEQ ID NO:10 and SEQ ID NO:11.

Preferably the aforementioned polypeptides are recombinantly produced.Most preferably the polypeptides according to the invention arepurified, and are substantially free of any other proteins orcontaminating host-originating material.

Further peptides of the present invention include isolated polypeptidesencoded by a polynucleotide comprising the sequence contained in SEQ IDNO:1.

The invention also provides an immunogenic fragment of a CASB7439polypeptide, that is a contiguous portion of the CASB7439 polypeptidewhich has the same or similar immunogenic properties to the polypeptidecomprising the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:7, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 or SEQ ID NO:14. That isto say, the fragment (if necessary when coupled to a carrier or as partof a larger fusion protein) is capable of raising an immune responsewhich recognises the CASB7439 polypeptide. Such an immunogenic fragmentmay include, for example, the CASB7439 polypeptide lacking an N-terminalleader sequence, a transmembrane domain or a C-terminal anchor domain.In a preferred aspect the immunogenic fragment of CASB7439 according tothe invention comprises substantially all of the extracellular domain ofa polypeptide which has at least 70% identity, preferably at least 80%identity, more preferably at least 90% identity, yet more preferably atleast 95% identity, most preferably at least 97-99% identity, to that ofSEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:11, SEQID NO:12 or SEQ ID NO:14 over the entire length of SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 or SEQ IDNO:14, respectively. Preferably an immunogenic fragment according to theinvention comprises at least one epitope.

Peptide fragments incorporating an epitope of CASB7439 typically willcomprise at least 7, preferably 9 or 10 contiguous amino acids from SEQID NO:2. Preferred epitopes are shown in SEQ ID NO:16 to SEQ ID NO:33.

Peptides that incorporate these epitopes form a preferred aspect of thepresent invention. Mimotopes which have the same characteristics asthese epitopes, and immunogens comprising such mimotopes which generatean immune response which cross-react with an epitope in the context ofthe CASB7439 molecule, also form part of the present invention.

The present invention, therefore, includes isolated peptidesencompassing these epitopes themselves, and any mimotope thereof. Themeaning of mimotope is defined as an entity which is sufficientlysimilar to the native CASB7439 epitope so as to be capable of beingrecognised by antibodies which recognise the native molecule; (Gheysen,H. M., et al., 1986, Synthetic peptides as antigens. Wiley, Chichester,Ciba foundation symposium 119, p 130-149; Gheysen, H. M., 1986,Molecular Immunology, 23,7, 709-715); or are capable of raisingantibodies, when coupled to a suitable carrier, which antibodiescross-react with the native molecule.

Peptide mimotopes of the above-identified epitopes may be designed for aparticular purpose by addition, deletion or substitution of electedamino acids. Thus, the peptides of the present invention may be modifiedfor the purposes of ease of conjugation to a protein carrier. Forexample, it may be desirable for some chemical conjugation methods toinclude a terminal cysteine to the epitope. In addition it may bedesirable for peptides conjugated to a protein carrier to include ahydrophobic terminus distal from the conjugated terminus of the peptide,such that the free unconjugated end of the peptide remains associatedwith the surface of the carrier protein. This reduces the conformationaldegrees of freedom of the peptide, and thus increases the probabilitythat the peptide is presented in a conformation which most closelyresembles that of the peptide as found in the context of the wholemolecule. For example, the peptides may be altered to have an N-terminalcysteine and a C-terminal hydrophobic amidated tail. Alternatively, theaddition or substitution of a D-stereoisomer form of one or more of theamino acids may be performed to create a beneficial derivative, forexample to enhance stability of the peptide. Those skilled in the artwill realise that such modified peptides, or mimotopes, could be awholly or partly non-peptide mimotope wherein the constituent residuesare not necessarily confined to the 20 naturally occurring amino acids.In addition, these may be cyclised by techniques known in the art toconstrain the peptide into a conformation that closely resembles itsshape when the peptide sequence is in the context of the whole molecule.A preferred method of cyclising a peptide comprises the addition of apair of cysteine residues to allow the formation of a disulphide bridge.

Further, those skilled in the art will realise that mimotopes orimmunogens of the present invention may be larger than theabove-identified epitopes, and as such may comprise the sequencesdisclosed herein. Accordingly, the mimotopes of the present inventionmay consist of addition of N and/or C terminal extensions of a number ofother natural residues at one or both ends. The peptide mimotopes mayalso be retro sequences of the natural sequences, in that the sequenceorientation is reversed; or alternatively the sequences may be entirelyor at least in part comprised of D-stereo isomer amino acids (inversosequences). Also, the peptide sequences may be retro-inverso incharacter, in that the sequence orientation is reversed and the aminoacids are of the D-stereoisomer form. Such retro or retro-inversopeptides have the advantage of being non-self, and as such may overcomeproblems of self-tolerance in the immune system.

Alternatively, peptide mimotopes may be identified using antibodieswhich are capable themselves of binding to the epitopes of the presentinvention using techniques such as phage display technology (EP 0 552267 B1). This technique, generates a large number of peptide sequenceswhich mimic the structure of the native peptides and are, therefore,capable of binding to anti-native peptide antibodies, but may notnecessarily themselves share significant sequence homology to the nativepeptide. This approach may have significant advantages by allowing thepossibility of identifying a peptide with enhanced immunogenicproperties, or may overcome any potential self-antigen toleranceproblems which may be associated with the use of the native peptidesequence. Additionally this technique allows the identification of arecognition pattern for each native-peptide in terms of its sharedchemical properties amongst recognised mimotope sequences.

The covalent coupling of the peptide to the immunogenic carrier can becarried out in a manner well known in the art. Thus, for example, fordirect covalent coupling it is possible to utilise a carbodiimide,glutaraldehyde or (N-[γ-maleimidobutyryloxy]succinimide ester, utilisingcommon commercially available heterobifunctional linkers such as CDAPand SPDP (using manufacturers instructions). After the couplingreaction, the immunogen can easily be isolated and purified by means ofa dialysis method, a gel filtration method, a fractionation method etc.

The types of carriers used in the immunogens of the present inventionwill be readily known to the man skilled in the art. The function of thecarrier is to provide cytokine help in order to help induce an immuneresponse against the peptide. A non-exhaustive list of carriers whichmay be used in the present invention include: Keyhole limpet Haemocyanin(KLH), serum albumins such as bovine serum albumin (BSA), inactivatedbacterial toxins such as tetanus or diptheria toxins (TT and DT), orrecombinant fragments thereof (for example, Domain 1 of Fragment C ofTT, or the translocation domain of DT), or the purified proteinderivative of tuberculin (PPD). Alternatively the mimotopes or epitopesmay be directly conjugated to liposome carriers, which may additionallycomprise immunogens capable of providing T-cell help. Preferably theratio of mimotopes to carrier is in the order of 1:1 to 20:1, andpreferably each carrier should carry between 3-15 peptides.

In an embodiment of the invention a preferred carrier is Protein D fromHaemophilus influenzae (EP 0 594 610 B1). Protein D is an IgD-bindingprotein from Haemophilus influenzae and has been patented by Forsgren(WO 91/18926, granted EP 0 594 610 B1). In some circumstances, forexample in recombinant immunogen expression systems it may be desirableto use fragments of protein D, for example Protein D ⅓^(rd) (comprisingthe N-terminal 100-110 amino acids of protein D (GB 9717953.5)).

Another preferred method of presenting the peptides of the presentinvention is in the context of a recombinant fusion molecule. Forexample, EP 0 421 635 B describes the use of chimaeric hepadnavirus coreantigen particles to present foreign peptide sequences in a virus-likeparticle. As such, immunogens of the present invention may comprisepeptides presented in chimaeric particles consisting of hepatitis B coreantigen. Additionally, the recombinant fusion proteins may comprise themimotopes of the present invention and a carrier protein, such as NS1 ofthe influenza virus. For any recombinantly expressed protein which formspart of the present invention, the nucleic acid which encodes saidimmunogen also forms an aspect of the present invention.

Peptides used in the present invention can be readily synthesised bysolid phase procedures well known in the art. Suitable syntheses may beperformed by utilising “T-boc” or “F-moc” procedures. Cyclic peptidescan be synthesised by the solid phase procedure employing the well-known“F-moc” procedure and polyamide resin in the fully automated apparatus.Alternatively, those skilled in the art will know the necessarylaboratory procedures to perform the process manually. Techniques andprocedures for solid phase synthesis are described in ‘Solid PhasePeptide Synthesis: A Practical Approach’ by E. Atherton and R. C.Sheppard, published by IRL at Oxford University Press (1989).Alternatively, the peptides may be produced by recombinant methods,including expressing nucleic acid molecules encoding the mimotopes in abacterial or mammalian cell line, followed by purification of theexpressed mimotope. Techniques for recombinant expression of peptidesand proteins are known in the art, and are described in Maniatis, T.,Fritsch, E. F. and Sambrook et al., Molecular cloning, a laboratorymanual, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1989).

In a further embodiment of the invention is provided a method ofproducing a polypeptide as described herein. The process of theinvention may be performed by conventional recombinant techniques suchas described in Maniatis et al., Molecular Cloning—A Laboratory Manual;Cold Spring Harbor, 1982-1989. Accordingly there is provided a processfor producing a polypeptide according to the invention, comprisingculturing a host cell under conditions sufficient for the production ofsaid polypeptide and recovering the polypeptide from the culture medium.In particular, the process of the invention may preferably comprise thesteps of:

-   -   i) preparing a replicable or integrating expression vector        capable, in a host cell, of expressing a DNA polymer comprising        a nucleotide sequence that encodes the protein or an immunogenic        derivative thereof;    -   ii) transforming a host cell with said vector;    -   ii) culturing said transformed host cell under conditions        permitting expression of said DNA polymer to produce said        protein; and    -   iv) recovering said protein.

The polypeptides or immunogenic fragment of the invention may be in theform of the “mature” protein or may be a part of a larger protein suchas a precursor or a fusion protein. It is often advantageous to includean additional amino acid sequence which contains secretory or leadersequences, pro-sequences, sequences which aid in purification such asmultiple histidine residues, or an additional sequence for stabilityduring recombinant production. Furthermore, addition of exogenouspolypeptide or lipid tail or polynucleotide sequences to increase theimmunogenic potential of the final molecule is also considered.

In one aspect, the invention relates to genetically engineered solublefusion proteins comprising a polypeptide of the present invention, or afragment thereof, and various portions of the constant regions of heavyor light chains of immunoglobulins of various subclasses (IgG, IgM, IgA,IgE). Preferred as an immunoglobulin is the constant part of the heavychain of human IgG, particularly IgG1, where fusion takes place at thehinge region. In a particular embodiment, the Fc part can be removedsimply by incorporation of a cleavage sequence which can be cleaved withblood clotting factor Xa.

Furthermore, this invention relates to processes for the preparation ofthese fusion proteins by genetic engineering, and to the use thereof fordrug screening, diagnosis and therapy. A particularly preferred aspectof the invention relates to the use of a polypeptide or a polynucleotidein the manufacture of a vaccine for immunotherapeutically treating apatient suffering from or susceptible to carcinoma, especially coloncancer or other colon-associated tumours or diseases. A further aspectof the invention also relates to polynucleotides encoding such fusionproteins. Examples of fusion protein technology can be found inInternational Patent Application Nos. WO94/29458 and WO94/22914.

The proteins may be chemically conjugated, or expressed as recombinantfusion proteins allowing increased levels to be produced in anexpression system as compared to non-fused protein. The fusion partnermay assist in providing T helper epitopes (immunological fusionpartner), preferably T helper epitopes recognised by humans, or assistin expressing the protein (expression enhancer) at higher yields thanthe native recombinant protein. Preferably the fusion partner will beboth an immunological fusion partner and expression enhancing partner.

Fusion partners include protein D from Haemophilus influenza B and thenon-structural protein from influenzae virus, NS1 (hemagglutinin).Another immunological fusion partner is the protein known as LYTA.Preferably the C terminal portion of the molecule is used. Lyta isderived from Streptococcus pneumoniae which synthesize anN-acetyl-L-alanine amidase, amidase LYTA, (coded by the lytA gene {Gene,43 (1986) page 265-272} an autolysin that specifically degrades certainbonds in the peptidoglycan backbone. The C-terminal domain of the LYTAprotein is responsible for the affinity to the choline or to somecholine analogues such as DEAE. This property has been exploited for thedevelopment of E. coli C-LYTA expressing plasmids useful for expressionof fusion proteins. Purification of hybrid proteins containing theC-LYTA fragment at its amino terminus has been described {Biotechnology:10, (1992) page 795-798}. It is possible to use the repeat portion ofthe Lyta molecule found in the C terminal end starting at residue 178,for example residues 188-305.

The present invention also includes xenogeneic forms (also termedortholog forms) of the aforementioned polypeptides, said xenogeneicforms referring to an antigen having substantial sequence identity tothe human antigen (also termed autologous antigen) which serves as areference antigen but which is derived from a different non-humanspecies. In this context the substantial identity refers to concordanceof an amino acid sequence with another amino acid sequence or of apolynucleotide sequence with another polynucleotide sequence when suchsequence are arranged in a best fit alignment in any of a number ofsequence alignment proteins known in the art. By substantial identity ismeant at least 70-95%, and preferably at least 85-95%, most preferablyat least 90%-95%, sequence identity between the compared sequences.Therefore according to the invention the xenogeneic CASB7439 polypeptidewill be a CASB7439 polypeptide which is xenogeneic with respect to humanCASB7439, in other words which is isolated from a species other thanhuman. In a preferred embodiment, the polypeptide is isolated frommouse, rat, pig, or rhesus monkey, most preferably from mouse or rat.Accordingly the present invention also provides a method of inducing animmune response against human CASB7439 having an amino acid sequence asset forth in any of the sequences SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:7,SEQ ID NO:10 or SEQ ID NO:11 in a human, comprising administering to thesubject an effective dosage of a composition comprising a xenogeneicform of said human CASB7439 as described herein. A preferred embodimentis a method of inducing an immune response against human CASB7439 usingthe xenogeneic CASB7439isolated from mouse, rat, pig or rhesus monkey.Another preferred method of inducing an immune response according to thepresent invention is using an antigen composition including a live viralexpression system which expresses said xenogeneic antigen. The preferredxenogeneic CASB7439 polypeptide has the sequence set forth in SEQ IDNO:12 (mouse) or in SEQ ID NO:14 (rat).

The isolated xenogeneic CASB7439 polypeptide will generally sharesubstantial sequence similarity, and include isolated polypeptidescomprising an amino acid sequence which has at least 70% identity,preferably at least 80% identity, more preferably at least 90% identity,yet more preferably at least 95% identity, most preferably at least97-99% identity, to that of SEQ ID NO:12 or SEQ ID NO:14 over the entirelength of SEQ ID NO:12 or SEQ ID NO:14. Accordingly the xenogeneicpolypeptide will comprise an immunogenic fragment of the polypeptide ofSEQ ID NO:12 or SEQ ID NO:14 in which the immunogenic activity of theimmunogenic fragment is substantially the same as the polypeptide of SEQID NO:12 or SEQ ID NO:14. In addition the xenogeneic CASB7439polypeptide can be a fragment of at least about 20 consecutive aminoacids, preferably about 30, more preferably about 50, yet morepreferably about 100, most preferably about 150 contiguous amino acidsselected from the amino acid sequences as shown in SEQ ID NO:12 or inSEQ ID NO:14. More particularly xenogeneic CASB7439 fragments willretain some functional property, preferably an immunological activity,of the larger molecule set forth in SEQ ID NO:12 or in SEQ ID NO14, andare useful in the methods described herein (e.g. in pharmaceutical andvaccine compositions, in diagnostics, etc.). In particular the fragmentswill be able to generate an immune response against the humancounterpart, such as the generation of cross-reactive antibodies whichreact with the autologous human form of CASB7439 as set forth in any ofthe SEQ ID NO: 2. In a specific embodiment, the xenogeneic polypeptideof the invention may be part of a larger fusion, comprising thexenogeneic CASB7439 polypeptide or fragment thereof and a heterologousprotein or part of a protein acting as a fusion partner as describedhereabove.

The present invention also includes variants of the aforementionedpolypeptides, that is polypeptides that vary from the referents byconservative amino acid substitutions, whereby a residue is substitutedby another with like characteristics. Typical such substitutions areamong Ala, Val, Leu and Ile; among Ser and Thr; among the acidicresidues Asp and Glu; among Asn and Gln; and among the basic residuesLys and Arg; or aromatic residues Phe and Tyr. Particularly preferredare variants in which several, 5-10, 1-5, 1-3, 1-2 or 1 amino acids aresubstituted, deleted, or added in any combination.

Polypeptides of the present invention can be prepared in any suitablemanner Such polypeptides include isolated naturally occurringpolypeptides, recombinantly produced polypeptides, syntheticallyproduced polypeptides, or polypeptides produced by a combination ofthese methods. Means for preparing such polypeptides are well understoodin the art.

In a further aspect, the present invention relates to CASB7439polynucleotides. Such polynucleotides include isolated polynucleotidescomprising a nucleotide sequence encoding a polypeptide which has atleast 70% identity, preferably at least 80% identity, more preferably atleast 90% identity, yet more preferably at least 95% identity, to theamino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:7, SEQ IDNO:10 or SEQ ID NO:11, over the entire length of SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:7, SEQ ID NO:10 or SEQ ID NO:11 respectively. In thisregard, encoded polypeptides which have at least 97% identity are highlypreferred, whilst those with at least 98-99% identity are more highlypreferred, and those with at least 99% identity are most highlypreferred.

Further polynucleotides of the present invention include isolatedpolynucleotides comprising a nucleotide sequence that has at least 70%identity, preferably at least 80% identity, more preferably at least 90%identity, yet more preferably at least 95% identity, to a nucleotidesequence encoding a polypeptide of SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:7, SEQ ID NO:10, or SEQ ID NO:11, over the entire coding region. Inthis regard, polynucleotides which have at least 97% identity are highlypreferred, whilst those with at least 98-99% identity are more highlypreferred, and those with at least 99% identity are most highlypreferred.

Further polynucleotides of the present invention include isolatedpolynucleotides comprising a nucleotide sequence which has at least 70%identity, preferably at least 80% identity, more preferably at least 90%identity, yet more preferably at least 95% identity, to SEQ ID NO:1, SEQID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:9, over theentire length of said sequences, or to the coding sequence of SEQ IDNO:1, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:9over the entire length of said coding sequence of SEQ ID NO:1, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:9. In thisregard, polynucleotides which have at least 97% identity are highlypreferred, whilst those with at least 98-99% identiy are more highlypreferred, and those with at least 99% identity are most highlypreferred. Such polynucleotides include a polynucleotide comprising thepolynucleotide of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6,SEQ ID NO:8 or SEQ ID NO:9 as well as the polynucleotide of SEQ ID NO:1,SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9 or thecoding region of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQID NO:8 or SEQ ID NO:9.

The present invention also provides a nucleic acid encoding theaforementioned xenogeneic proteins of the present invention and theiruse in medicine. In a preferred embodiment, the xenogeneic CASB7439polynucleotide for use in pharmaceutical compositions has the sequenceset forth in SEQ ID NO:13 (mouse) or in SEQ ID NO:15 (rat). The isolatedxenogeneic CASB7439 polynucleotides according to the invention may besingle-stranded (coding or antisense) or double-stranded, and may be DNA(genomic, cDNA or synthetic) or RNA molecules. Additional coding ornon-coding sequences may, but need not, be present within apolynucleotide of the present invention. In other related embodiments,the present invention provides polynucleotide variants havingsubstantial identity to the sequences disclosed herein in SEQ ID NO:13or in SEQ ID NO:15, for example those comprising at least 70% sequenceidentity, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or99% or higher, sequence identity compared to a polynucleotide sequenceof this invention using the methods described herein, (e.g., BLASTanalysis using standard parameters). In a related embodiment, theisolated xenogeneic polynucleotide of the invention will comprise anucleotide sequence encoding a polypeptide that has at least 90%,preferably 95% and above, identity to the amino acid sequence of SEQ IDNO:12 or of SEQ ID NO:14, over the entire length of SEQ ID NO:12 or ofSEQ ID NO:14; or a nucleotide sequence complementary to said isolatedpolynucleotide.

The invention also provides polynucleotides which are complementary toall the above described polynucleotides.

Said polynucleotides can be inserted in a suitable plasmid, recombinantmicroorganism vector or a recombinant live microorganism and used forimmunization (see for example Wolff et. al., Science 247:1465-1468(1990); Corr et. al., J. Exp. Med. 184:1555-1560 (1996); Doe et. al.,Proc. Natl. Acad. Sci. 93:8578-8583 (1996)). Accordingly there isprovided in the present invention an expression vector or recombinantlive microorganism comprising said polynucleotides as hereabove defined.

The invention also provides a fragment of a CASB7439 polynucleotidewhich when administered to a subject has the same immunogenic propertiesas the polynucleotide of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:13 or SEQ ID NO:15.

The invention also provides a polynucleotide encoding an immunologicalfragment of a CASB7439 polypeptide as hereinbefore defined.

The fragments have a level of immunogenic activity of at least about50%, preferably at least about 70% and more preferably at least about90% of the level of immunogenic activity of a polypeptide sequence setforth in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:10 or SEQ IDNO:11, SEQ ID NO:12 or SEQ ID NO:14 or a polypeptide sequence encoded bya polynucleotide sequence set forth in SEQ ID NO:1, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:13 or SEQ IDNO:15.

The polypeptide fragments according to the invention preferably compriseat least about 5, 10, 15, 20, 25, 50, or 100 contiguous amino acids, ormore, including all intermediate lengths, of a polypeptide compositionset forth herein, such as those set forth in SEQ ID NO:2, SEQ ID NO:3,SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 or SEQ ID NO:14,or those encoded by a polynucleotide sequence set forth in a sequence ofSEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:9, SEQ ID NO:13 or SEQ ID NO:15.

The nucleotide sequence of SEQ ID NO:1 is a cDNA sequence whichcomprises a polypeptide encoding sequence (nucleotide 545 to 1126)encoding a polypeptide of 193 amino acids, the polypeptide of SEQ IDNO:2. The nucleotide sequence encoding the polypeptide of SEQ ID NO:2may be identical to the polypeptide encoding sequence contained in SEQID NO:1 or it may be a sequence other than the one contained in SEQ IDNO:1, which, as a result of the redundancy (degeneracy) of the geneticcode, also encodes the polypeptide of SEQ ID NO:2. The polypeptide ofthe SEQ ID NO:2 is structurally related to other proteins of the achaetescute family, and is also named “human Achaete Scute homologue 2”(HASH2) (accession number NP_(—)005161 and AAB86993).

Human Achaete Scute homologue 2 (HASH2) gene, officially designatedhuman ASCL2 (Achaete Scute complex like 2) is a homologue of theDrosophila Achaete and Scute genes. Human ASCL2 is expressed in theextravillus trophoblasts of the developing placenta only, and maps onchromosome 11p15 close to IGF2 and H19. The mouse achaete-scutehomolog-2 gene (MASH2) encodes a transcription factor playing a role inthe development of the trophoblast. The Mash2 gene is paternallyimprinted in the mouse, and the lack of human ASCL2 expression innon-malignant hydatidiform (androgenetic) moles indicates that humanAscl2 is also imprinted in man.

Ascl2 genes are members of the basic helix-loop-helix (BHLH) family oftranscription factors. They activate transcription by binding to the Ebox (5′-CANNTG-3′). Dimerization with other BHLH proteins is requiredfor efficient DNA binding. They are involved in the determination of theneuronal precursors in the peripheral nervous system and the centralnervous system in drosophila melanogaster, and probably in mammals aswell.

The complementary strand of the nucleotide sequence of SEQ ID NO:1 isthe polynucleotide sequence of SEQ ID NO:6. This strand also comprisestwo other polypeptide encoding sequences. The first polypeptide encodingsequence (nucleotide 1184 to 399 of SEQ ID:1, nucleotide 608 to 1393 ofSEQ ID NO:6) encodes a polypeptide of 262 amino acids, the polypeptideof SEQ ID NO:3. The second polypeptide encoding sequence (nucleotide 840to 262 of SEQ ID NO:1, nucleotide 952 to 1530 of SEQ ID NO:6) encodes apolypeptide of 193 amino acids, the polypeptide of SEQ ID NO: 11. Thenucleotide sequence encoding the polypeptides of SEQ ID NO:3 and SEQ IDNO:11 may be identical to the polypeptides encoding sequence containedin SEQ ID NO: 6 or it may be a sequence other than the one contained inSEQ ID NO: 6, which, as a result of the redundancy (degeneracy) of thegenetic code, also encodes the polypeptides of SEQ ID NO:3 and 11. Thepolypeptide of the SEQ ID NO:3 is structurally related to other proteinsof the splicing coactivator protein family, having homology and/orstructural similarity with homo sapiens splicing coactivator subunitsrm300 (genbank accession AAF21439). The polypeptide of SEQ ID NO:11 isnot related to any known protein. Polypeptide sequences as set forth inSEQ ID NO:3 and SEQ ID NO:11, and polynucleotide sequences as set forthin SEQ ID NO:6 are novel and also form part of the invention.

Preferred polypeptides and polynucleotides of the present invention areexpected to have, inter alia, similar biological functions/properties totheir homologous polypeptides and polynucleotides. Furthermore,preferred polypeptides, immunological fragments and polynucleotides ofthe present invention have at least one activity of either SEQ ID NO:1,SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:11 as appropriate.

The present invention also relates to partial or other incompletepolynucleotide and polypeptide sequences which were first identifiedprior to the determination of the corresponding full length sequences ofSEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:11.

Accordingly, in a further aspect, the present invention provides for anisolated polynucleotide which:

(a) comprises a nucleotide sequence which has at least 70% identity,preferably at least 80% identity, more preferably at least 90% identity,yet more preferably at least 95% identity, even more preferably at least97-99% identity to SEQ ID NO:4 and 5 over the entire length of SEQ ID NO4 and 5;

(b) has a nucleotide sequence which has at least 70% identity,preferably at least 80% identity, more preferably at least 90% identity,yet more preferably at least 95% identity, even more preferably at least97-99% identity, to SEQ ID NO:1 or SEQ ID NO:6 over the entire length ofSEQ ID NO:4 and SEQ ID NO:5 respectively;

(c) the polynucleotide of SEQ ID NO:4 and SEQ ID NO:5; or

(d) a nucleotide sequence encoding a polypeptide which has at least 70%identity, preferably at least 80% identity, more preferably at least 90%identity, yet more preferably at least 95% identity, even morepreferably at least 97-99% identity, to the amino acid sequence of SEQID NO:2 and SEQ ID NO:7 respectively, over the entire length of SEQ IDNO:2 and 7, as well as the polynucleotides of SEQ ID NO:4 and 5.

The present invention further provides for a polypeptide which:

(a) comprises an amino acid sequence which has at least 70% identity,preferably at least 80% identity, more preferably at least 90% identity,yet more preferably at least 95% identity, most preferably at least97-99% identity, to that of SEQ ID NO:2 and 7 over the entire length ofSEQ ID NO:2 or 7;

(b) has an amino acid sequence which is at least 70% identity,preferably at least 80% identity, more preferably at least 90% identity,yet more preferably at least 95% identity, most preferably at least97-99% identity, to the amino acid sequence of SEQ ID NO:2 or 7 over theentire length of SEQ ID NO:2 or 7;

(c) comprises the amino acid of SEQ ID NO:2 or 7; and

(d) is the polypeptide of SEQ ID NO: 7;

as well as polypeptides encoded by a polynucleotide comprising thesequence contained in SEQ ID NO:4 and 5.

Polynucleotides of the present invention may be obtained, using standardcloning and screening techniques, from a cDNA library derived from mRNAin cells of human colon cancer, (for example Sambrook et al., MolecularCloning: A Laboratory Manual, 2^(nd) Ed., Cold Spring harbor LaboratoryPress, Cold Spring harbor, N.Y. (1989)). Polynucleotides of theinvention can also be obtained from natural sources such as genomic DNAlibraries or can be synthesized using well known and commerciallyavailable techniques.

When polynucleotides of the present invention are used for therecombinant production of polypeptides of the present invention, thepolynucleotide may include the coding sequence for the maturepolypeptide, by itself; or the coding sequence for the maturepolypeptide in reading frame with other coding sequences, such as thoseencoding a leader or secretory sequence, a pre-, or pro- orprepro-protein sequence, or other fusion peptide portions. For example,a marker sequence which facilitates purification of the fusedpolypeptide can be encoded. In certain preferred embodiments of thisaspect of the invention, the marker sequence is a hexa-histidinepeptide, as provided in the pQE vector (Qiagen, Inc.) and described inGentz et al., Proc Natl Acad Sci USA (1989) 86:821-824, or is an HA tag.The polynucleotide may also contain non-coding 5′ and 3′ sequences, suchas transcribed, non-translated sequences, splicing and polyadenylationsignals, ribosome binding sites and sequences that stabilize mRNA.

Further embodiments of the present invention include polynucleotidesencoding polypeptide variants which comprise the amino acid sequence ofSEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:13 or SEQID NO:15 and in which several, for instance from 5 to 10, 1 to 5, 1 to3, 1 to 2 or 1, amino acid residues are substituted, deleted or added,in any combination.

Polynucleotides which are identical or sufficiently identical to anucleotide sequence contained in SEQ ID NO:1 or in SEQ ID NO:6, may beused as hybridization probes for cDNA and genomic DNA or as primers fora nucleic acid amplification (PCR) reaction, to isolate full-lengthcDNAs and genomic clones encoding polypeptides of the present inventionand to isolate cDNA and genomic clones of other genes (including genesencoding paralogs from human sources and orthologs and paralogs fromspecies other than human) that have a high sequence similarity to SEQ IDNO:1 or to SEQ ID NO:6. Typically these nucleotide sequences are 70%identical, preferably 80% identical, more preferably 90% identical, mostpreferably 95% identical to that of the referent. The probes or primerswill generally comprise at least 15 nucleotides, preferably, at least 30nucleotides and may have at least 50 nucleotides. Particularly preferredprobes will have between 30 and 50 nucleotides. Particularly preferredprimers will have between 20 and 25 nucleotides. In particular,polypeptides or polynucleotides derived from sequences from homologousanimal origin could be used as immunogens to obtain a cross-reactiveimmune response to the human gene.

A polynucleotide encoding a polypeptide of the present invention,including homologs from species other than human, may be obtained by aprocess which comprises the steps of screening an appropriate libraryunder stringent hybridization conditions with a labeled probe having thesequence of SEQ ID NO: 1 or SEQ ID NO:6 or a fragment thereof; andisolating full-length cDNA and genomic clones containing saidpolynucleotide sequence. Such hybridization techniques are well known tothe skilled artisan. Preferred stringent hybridization conditionsinclude overnight incubation at 42° C. in a solution comprising: 50%formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodiumphosphate (pH7.6), 5× Denhardt's solution, 10% dextran sulfate, and 20microgram/ml denatured, sheared salmon sperm DNA; followed by washingthe filters in 0.1×SSC at about 65° C. Thus the present invention alsoincludes polynucleotides obtainable by screening an appropriate libraryunder stingent hybridization conditions with a labeled probe having thesequence of SEQ ID NO:1 or SEQ ID NO:6 or a fragment thereof.

The skilled artisan will appreciate that, in many cases, an isolatedcDNA sequence will be incomplete, in that the region coding for thepolypeptide is short at the 5′ end of the cDNA.

There are several methods available and well known to those skilled inthe art to obtain full-length cDNAs, or extend short cDNAs, for examplethose based on the method of Rapid Amplification of cDNA ends (RACE)(see, for example, Frohman et al., PNAS USA 85, 8998-9002, 1988). Recentmodifications of the technique, exemplified by the Marathon™ technology(Clontech Laboratories Inc.) for example, have significantly simplifiedthe search for longer cDNAs. In the Marathon™ technology, cDNAs havebeen prepared from mRNA extracted from a chosen tissue and an ‘adaptor’sequence ligated onto each end. Nucleic acid amplification (PCR) is thencarried out to amplify the ‘missing’ 5′ end of the cDNA using acombination of gene specific and adaptor specific oligonucleotideprimers. The PCR reaction is then repeated using ‘nested’ primers, thatis, primers designed to anneal within the amplified product (typicallyan adaptor specific primer that anneals further 3′ in the adaptorsequence and a gene specific primer that anneals further 5′ in the knowngene sequence). The products of this reaction can then be analysed byDNA sequencing and a full-length cDNA constructed either by joining theproduct directly to the existing cDNA to give a complete sequence, orcarrying out a separate full-length PCR using the new sequenceinformation for the design of the 5′ primer.

Recombinant polypeptides of the present invention may be prepared byprocesses well known in the art from genetically engineered host cellscomprising expression systems. Accordingly, in a further aspect, thepresent invention relates to an expression system which comprises apolynucleotide of the present invention, to host cells which aregenetically engineered with such expression systems and to theproduction of polypeptides of the invention by recombinant techniques.Cell-free translation systems can also be employed to produce suchproteins using RNAs derived from the DNA constructs of the presentinvention.

For recombinant production, host cells can be genetically engineered toincorporate expression systems or portions thereof for polynucleotidesof the present invention. Introduction of polynucleotides into hostcells can be effected by methods described in many standard laboratorymanuals, such as Davis et al., Basic Methods in Molecular Biology (1986)and Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed.,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).Preferred such methods include, for instance, calcium phosphatetransfection, DEAE-dextran mediated transfection, transvection,microinjection, cationic lipid-mediated transfection, electroporation,transduction, scrape loading, ballistic introduction or infection.

Preferably the proteins of the invention are coexpressed withthioredoxin in trans (TIT). Coexpression of thioredoxin in trans versusin cis is preferred to keep antigen free of thioredoxin without the needfor protease. Thioredoxin coexpression eases the solubilisation of theproteins of the invention. Thioredoxin coexpression has also asignificant impact on protein purification yield, on purified-proteinsolubility and quality.

Representative examples of appropriate hosts include bacterial cells,such as Streptococci, Staphylococci, E. coli, Streptomyces and Bacillussubtilis cells; fungal cells, such as yeast cells and Aspergillus cells;insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animalcells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanomacells; and plant cells.

A great variety of expression systems can be used, for instance,chromosomal, episomal and virus-derived systems, e.g., vectors derivedfrom bacterial plasmids, from bacteriophage, from transposons, fromyeast episomes, from insertion elements, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such asSV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses and retroviruses, and vectors derived from combinations thereof,such as those derived from plasmid and bacteriophage genetic elements,such as cosmids and phagemids. The expression systems may containcontrol regions that regulate as well as engender expression. Generally,any system or vector which is able to maintain, propagate or express apolynucleotide to produce a polypeptide in a host may be used. Theappropriate nucleotide sequence may be inserted into an expressionsystem by any of a variety of well-known and routine techniques, suchas, for example, those set forth in Sambrook et al., Molecular Cloning,A Laboratory Manual (supra). Appropriate secretion signals may beincorporated into the desired polypeptide to allow secretion of thetranslated protein into the lumen of the endoplasmic reticulum, theperiplasmic space or the extracellular environment. These signals may beendogenous to the polypeptide or they may be heterologous signals.

The expression system may also be a recombinant live microorganism, suchas a virus or bacterium. The gene of interest can be inserted into thegenome of a live recombinant virus or bacterium. Inoculation and in vivoinfection with this live vector will lead to in vivo expression of theantigen and induction of immune responses.

Therefore, in certain embodiments, polynucleotides encoding immunogenicpolypeptides of the present invention are introduced into suitablemammalian host cells for expression using any of a number of knownviral-based systems. In one illustrative embodiment, retrovirusesprovide a convenient and effective platform for gene delivery systems. Aselected nucleotide sequence encoding a polypeptide of the presentinvention can be inserted into a vector and packaged in retroviralparticles using techniques known in the art. The recombinant virus canthen be isolated and delivered to a subject. A number of illustrativeretroviral systems have been described (e.g., U.S. Pat. No. 5,219,740;Miller and Rosman (1989) BioTechniques 7:980-990; Miller, A. D. (1990)Human Gene Therapy 1:5-14; Scarpa et al. (1991) Virology 180:849-852;Burns et al. (1993) Proc. Natl. Acad. Sci. USA 90:8033-8037; andBoris-Lawrie and Temin (1993) Cur. Opin. Genet. Develop. 3:102-109.

In addition, a number of illustrative adenovirus-based systems have alsobeen described. Unlike retroviruses which integrate into the hostgenome, adenoviruses persist extrachromosomally thus minimizing therisks associated with insertional mutagenesis (Haj-Ahmad and Graham(1986) J. Virol. 57:267-274; Bett et al. (1993) J. Virol. 67:5911-5921;Mittereder et al. (1994) Human Gene Therapy 5:717-729; Seth et al.(1994) J. Virol. 68:933-940; Barr et al. (1994) Gene Therapy 1:51-58;Berkner, K. L. (1988) BioTechniques 6:616-629; and Rich et al. (1993)Human Gene Therapy 4:461-476).

Various adeno-associated virus (AAV) vector systems have also beendeveloped for polynucleotide delivery. AAV vectors can be readilyconstructed using techniques well known in the art. See, e.g., U.S. Pat.Nos. 5,173,414 and 5,139,941; International Publication Nos. WO 92/01070and WO 93/03769; Lebkowski et al. (1988) Molec. Cell. Biol. 8:3988-3996;Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press);Carter, B. J. (1992) Current Opinion in Biotechnology 3:533-539;Muzyczka, N. (1992) Current Topics in Microbiol. and Immunol.158:97-129; Kotin, R. M. (1994) Human Gene Therapy 5:793-801; Shellingand Smith (1994) Gene Therapy 1:165-169; and Zhou et al. (1994) J. Exp.Med. 179:1867-1875.

Additional viral vectors useful for delivering the nucleic acidmolecules encoding polypeptides of the present invention by genetransfer include those derived from the pox family of viruses, such asvaccinia virus and avian poxvirus. By way of example, vaccinia virusrecombinants expressing the novel molecules can be constructed asfollows. The DNA encoding a polypeptide is first inserted into anappropriate vector so that it is adjacent to a vaccinia promoter andflanking vaccinia DNA sequences, such as the sequence encoding thymidinekinase (TK). This vector is then used to transfect cells which aresimultaneously infected with vaccinia. Homologous recombination servesto insert the vaccinia promoter plus the gene encoding the polypeptideof interest into the viral genome. The resulting TK.sup.(−) recombinantcan be selected by culturing the cells in the presence of5-bromodeoxyuridine and picking viral plaques resistant thereto.

A vaccinia-based infection/transfection system can be conveniently usedto provide for inducible, transient expression or coexpression of one ormore polypeptides described herein in host cells of an organism. In thisparticular system, cells are first infected in vitro with a vacciniavirus recombinant that encodes the bacteriophage T7 RNA polymerase. Thispolymerase displays exquisite specificity in that it only transcribestemplates bearing T7 promoters. Following infection, cells aretransfected with the polynucleotide or polynucleotides of interest,driven by a T7 promoter. The polymerase expressed in the cytoplasm fromthe vaccinia virus recombinant transcribes the transfected DNA into RNAwhich is then translated into polypeptide by the host translationalmachinery. The method provides for high level, transient, cytoplasmicproduction of large quantities of RNA and its translation products. See,e.g., Elroy-Stein and Moss, Proc. Natl. Acad. Sci. USA (1990)87:6743-6747; Fuerst et al. Proc. Natl. Acad. Sci. USA (1986)83:8122-8126.

Alternatively, avipoxviruses, such as the fowlpox and canarypox viruses,can also be used to deliver the coding sequences of interest.Recombinant avipox viruses, expressing immunogens from mammalianpathogens, are known to confer protective immunity when administered tonon-avian species. The use of an Avipox vector is particularly desirablein human and other mammalian species since members of the Avipox genuscan only productively replicate in susceptible avian species andtherefore are not infective in mammalian cells. Methods for producingrecombinant Avipoxviruses are known in the art and employ geneticrecombination, as described above with respect to the production ofvaccinia viruses. See, e.g., WO 91/12882; WO 89/03429; and WO 92/03545.

Any of a number of alphavirus vectors can also be used for delivery ofpolynucleotide compositions of the present invention, such as thosevectors described in U.S. Pat. Nos. 5,843,723; 6,015,686; 6,008,035 and6,015,694. Certain vectors based on Venezuelan Equine Encephalitis (VEE)can also be used, illustrative examples of which can be found in U.S.Pat. Nos. 5,505,947 and 5,643,576.

Moreover, molecular conjugate vectors, such as the adenovirus chimericvectors described in Michael et al. J. Biol. Chem. (1993) 268:6866-6869and Wagner et al. Proc. Natl. Acad. Sci. USA (1992) 89:6099-6103, canalso be used for gene delivery under the invention.

Additional illustrative information on these and other known viral-baseddelivery systems can be found, for example, in Fisher-Hoch et al., Proc.Natl. Acad. Sci. USA 86:317-321, 1989; Flexner et al., Ann. N.Y. Acad.Sci. 569:86-103, 1989; Flexner et al., Vaccine 8:17-21, 1990; U.S. Pat.Nos. 4,603,112, 4,769,330, and 5,017,487; WO 89/01973; U.S. Pat. No.4,777,127; GB 2,200,651; EP 0,345,242; WO 91/02805; Berkner,Biotechniques 6:616-627, 1988; Rosenfeld et al., Science 252:431-434,1991; Kolls et al., Proc. Natl. Acad. Sci. USA 91:215-219, 1994;Kass-Eisler et al., Proc. Natl. Acad. Sci. USA 90:11498-11502, 1993;Guzman et al., Circulation 88:2838-2848, 1993; and Guzman et al., Cir.Res. 73:1202-1207, 1993.

The recombinant live microorganisms described above can be virulent, orattenuated in various ways in order to obtain live vaccines. Such livevaccines also form part of the invention.

In certain embodiments, a polynucleotide may be integrated into thegenome of a target cell. This integration may be in the specificlocation and orientation via homologous recombination (gene replacement)or it may be integrated in a random, non-specific location (geneaugmentation). In yet further embodiments, the polynucleotide may bestably maintained in the cell as a separate, episomal segment of DNA.Such polynucleotide segments or “episomes” encode sequences sufficientto permit maintenance and replication independent of or insynchronization with the host cell cycle. The manner in which theexpression construct is delivered to a cell and where in the cell thepolynucleotide remains is dependent on the type of expression constructemployed.

In another embodiment of the invention, a polynucleotide isadministered/delivered as “naked” DNA, for example as described in Ulmeret al., Science 259:1745-1749, 1993 and reviewed by Cohen, Science259:1691-1692, 1993. The uptake of naked DNA may be increased by coatingthe DNA onto biodegradable beads, which are efficiently transported intothe cells.

In still another embodiment, a composition of the present invention canbe delivered via a particle bombardment approach, many of which havebeen described. In one illustrative example, gas-driven particleacceleration can be achieved with devices such as those manufactured byPowderject Pharmaceuticals PLC (Oxford, UK) and Powderject Vaccines Inc.(Madison, Wis.), some examples of which are described in U.S. Pat. Nos.5,846,796; 6,010,478; 5,865,796; 5,584,807; and EP Patent No. 0500 799.This approach offers a needle-free delivery approach wherein a drypowder formulation of microscopic particles, such as polynucleotide orpolypeptide particles, are accelerated to high speed within a helium gasjet generated by a hand held device, propelling the particles into atarget tissue of interest.

In a related embodiment, other devices and methods that may be usefulfor gas-driven needle-less injection of compositions of the presentinvention include those provided by Bioject, Inc. (Portland, Oreg.),some examples of which are described in U.S. Pat. Nos. 4,790,824;5,064,413; 5,312,335; 5,383,851; 5,399,163; 5,520,639 and 5,993,412.

Polypeptides of the present invention can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. Most preferably, ion metalaffinity chromatography (IMAC) is employed for purification. Well knowntechniques for refolding proteins may be employed to regenerate activeconformation when the polypeptide is denatured during intracellularsynthesis, isolation and or purification.

Another important aspect of the invention relates to a method forinducing, re-inforcing or modulating an immunological response in amammal which comprises inoculating the mammal with a fragment or theentire polypeptide or polynucleotide of the invention, adequate toproduce antibody and/or T cell immune response for immunoprophylaxis orfor therapeutic treatment of cancer, more particularly colorectalcancer, and autoimmune disease and related conditions. Yet anotheraspect of the invention relates to a method of inducing, re-inforcing ormodulating immunological response in a mammal which comprises,delivering a polypeptide of the present invention via a vector or celldirecting expression of the polynucleotide and coding for thepolypeptide in vivo in order to induce such an immunological response toproduce immune responses for prophylaxis or treatment of said mammalfrom diseases.

A further aspect of the invention relates to an immunological/vaccineformulation (composition) and to their use in medicine. Thesecompositions, when introduced into a mammalian host, induce, re-inforceor modulate an immunological response in that mammal to a polypeptide ofthe present invention wherein the composition comprises a polypeptide orpolynucleotide of the invention or an immunological fragment thereof asherein before defined. More particularly the immunogenic compositionaccording to the present invention comprises a safe and effective amountof a CASB7439 polypeptide, or immunogenic fragment thereof wherein theCASB7439 polypeptide is selected from the group comprising SEQ ID NO:2,SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 orSEQ ID NO:14. In another embodiment, the imunogenic compositioncomprises a safe and effective amount of a CASB7439-encodingpolynucleotide, or fragment thereof wherein the CASB7439-encodingpolynucleotide is selected from the group comprising SEQ ID NO:1, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:13or SEQ ID NO:15.

The vaccine formulation according to the invention may further comprisea suitable, i.e. pharmaceutically acceptable carrier. Since apolypeptide may be broken down in the stomach, it is preferablyadministered parenterally (for instance, subcutaneous, intramuscular,intravenous, or intradermal injection). Formulations suitable forparenteral administration include aqueous and non-aqueous sterileinjection solutions which may contain anti-oxidants, buffers,bacteriostats and solutes which render the formulation isotonic with theblood of the recipient; and aqueous and non-aqueous sterile suspensionswhich may include suspending agents or thickening agents. Theformulations may be presented in unit-dose or multi-dose containers, forexample, sealed ampoules and vials and may be stored in a freeze-driedcondition requiring only the addition of the sterile liquid carrierimmediately prior to use.

A further aspect of the invention relates to the in vitro induction ofimmune responses to a fragment or the entire polypeptide orpolynucleotide of the present invention or a molecule comprising thepolypeptide or polynucleotide of the present invention, using cells fromthe immune system of a mammal, and reinfusing these activated immunecells of the mammal for the treatment of disease. Activation of thecells from the immune system is achieved by in vitro incubation with theentire polypeptide or polynucleotide of the present invention or amolecule comprising the polypeptide or polynucleotide of the presentinvention in the presence or absence of various immunomodulatormolecules. A further aspect of the invention relates to the immunizationof a mammal by administration of antigen presenting cells modified by invitro loading with part or the entire polypeptide of the presentinvention or a molecule comprising the polypeptide of the presentinvention and administered in vivo in an immunogenic way. Alternatively,antigen presenting cells can be transfected in vitro with a vectorcontaining a fragment or the entire polynucleotide of the presentinvention or a molecule comprising the polynucleotide of the presentinvention, such as to express the corresponding polypeptide, andadministered in vivo in an immunogenic way. Accordingly, thepharmaceutical compositions of the invention will comprise an effectiveamount of antigen presenting cells, modified by in vitro loading with aCASB7439 polypeptide, or genetically modified in vitro to express aCASB7439 polypeptide and a pharmaceutically effective carrier.

According to another embodiment, the pharmaceutical/immunogeniccompositions described herein will comprise one or more immunostimulantsin addition to the immunogenic polynucleotide, polypeptide, antibody,T-cell and/or antigen presenting cell (APC) compositions of thisinvention. Accordingly there is herein provided a process for theproduction of said immunogenic composition, comprising admixing aCASB7439 polypeptide or a CASB7439-encoding polynucleotide with asuitable adjuvant/immunostimulant, diluent or other pharmaceuticallyacceptable carrier. An immunostimulant refers to essentially anysubstance that enhances or potentiates an immune response (antibodyand/or cell-mediated) to an exogenous antigen. One preferred type ofimmunostimulant comprises an adjuvant. Many adjuvants contain asubstance designed to protect the antigen from rapid catabolism, such asaluminum hydroxide or mineral oil, and a stimulator of immune responses,such as lipid A, Bortadella pertussis or Mycobacterium tuberculosisderived proteins. Certain adjuvants are commercially available as, forexample, Freund's Incomplete Adjuvant and Complete Adjuvant (DifcoLaboratories, Detroit, Mich.); Merck Adjuvant 65 (Merck and Company,Inc., Rahway, N.J.); AS-2 (SmithKline Beecham, Philadelphia, Pa.);aluminum salts such as aluminum hydroxide gel (alum) or aluminumphosphate; salts of calcium, iron or zinc; an insoluble suspension ofacylated tyrosine; acylated sugars; cationically or anionicallyderivatized polysaccharides; polyphosphazenes; biodegradablemicrospheres; monophosphoryl lipid A and quil A. Cytokines, such asGM-CSF, interleukin-2, -7, -12, and other like growth factors, may alsobe used as adjuvants.

Within certain embodiments of the invention, the adjuvant composition ispreferably one that induces an immune response predominantly of the Th1type. High levels of Th1-type cytokines (e.g., IFN-γ, TNFα, IL-2 andIL-12) tend to favor the induction of cell mediated immune responses toan administered antigen. In contrast, high levels of Th2-type cytokines(e.g., IL-4, IL-5, IL-6 and IL-10) tend to favor the induction ofhumoral immune responses. Following application of a vaccine as providedherein, a patient will support an immune response that includes Th1- andTh2-type responses. Within a preferred embodiment, in which a responseis predominantly Th1-type, the level of Th1-type cytokines will increaseto a greater extent than the level of Th2-type cytokines. The levels ofthese cytokines may be readily assessed using standard assays. For areview of the families of cytokines, see Mosmann and Coffman, Ann. Rev.Immunol. 7:145-173, 1989.

Certain preferred adjuvants for eliciting a predominantly Th1-typeresponse include, for example, a combination of monophosphoryl lipid A,preferably 3-de-O-acylated monophosphoryl lipid A, together with analuminum salt. MPL® adjuvants are available from Corixa Corporation(Seattle, Wash.; see, for example, U.S. Pat. Nos. 4,436,727; 4,877,611;4,866,034 and 4,912,094). CpG-containing oligonucleotides (in which theCpG dinucleotide is unmethylated) also induce a predominantly Th1response. Such oligonucleotides are well known and are described, forexample, in WO 96/02555, WO 99/33488 and U.S. Pat. Nos. 6,008,200 and5,856,462 Immunostimulatory DNA sequences are also described, forexample, by Sato et al., Science 273:352, 1996. Another preferredadjuvant comprises a saponin, such as Quil A, or derivatives thereof,including QS21 and QS7 (Aquila Biopharmaceuticals Inc., Framingham,Mass.); Escin; Digitonin; or Gypsophila or Chenopodium quinoa saponins.Other preferred formulations include more than one saponin in theadjuvant combinations of the present invention, for example combinationsof at least two of the following group comprising QS21, QS7, Quil A,β-escin, or digitonin.

Alternatively the saponin formulations may be combined with vaccinevehicles composed of chitosan or other polycationic polymers,polylactide and polylactide-co-glycolide particles, poly-N-acetylglucosamine-based polymer matrix, particles composed of polysaccharidesor chemically modified polysaccharides, liposomes and lipid-basedparticles, particles composed of glycerol monoesters, etc. The saponinsmay also be formulated in the presence of cholesterol to formparticulate structures such as liposomes or ISCOMs. Furthermore, thesaponins may be formulated together with a polyoxyethylene ether orester, in either a non-particulate solution or suspension, or in aparticulate structure such as a paucilamelar liposome or ISCOM. Thesaponins may also be formulated with excipients such as Carbopol^(R) toincrease viscosity, or may be formulated in a dry powder form with apowder excipient such as lactose.

In one preferred embodiment, the adjuvant system includes thecombination of a monophosphoryl lipid A and a saponin derivative, suchas the combination of QS21 and 3D-MPL® adjuvant, as described in WO94/00153, or a less reactogenic composition where the QS21 is quenchedwith cholesterol, as described in WO 96/33739. Other preferredformulations comprise an oil-in-water emulsion and tocopherol. Anotherparticularly preferred adjuvant formulation employing QS21, 3D-MPL®adjuvant and tocopherol in an oil-in-water emulsion is described in WO95/17210.

Another enhanced adjuvant system involves the combination of aCpG-containing oligonucleotide and a saponin derivative particularly thecombination of CpG and QS21 as disclosed in WO 00/09159. Preferably theformulation additionally comprises an oil in water emulsion andtocopherol.

Additional illustrative adjuvants for use in the pharmaceuticalcompositions of the invention include Montanide ISA 720 (Seppic,France), SAF (Chiron, Calif., United States), ISCOMS (CSL), MF-59(Chiron), the SBAS series of adjuvants (e.g., SBAS-2 or SBAS-4,available from SmithKline Beecham, Rixensart, Belgium), Detox(Enhanzyn®) (Corixa, Hamilton, Mont.), RC-529 (Corixa, Hamilton, Mont.)and other aminoalkyl glucosaminide 4-phosphates (AGPs), such as thosedescribed in pending U.S. patent application Ser. Nos. 08/853,826 and09/074,720, the disclosures of which are incorporated herein byreference in their entireties, and polyoxyethylene ether adjuvants suchas those described in WO 99/52549A1.

-   -   Other preferred adjuvants include adjuvant molecules of the        general formula (I):

HO(CH₂CH₂O)_(n)-A-R

-   -   Wherein, n is 1-50, A is a bond or —C(O)—, R is C₁₋₅₀ alkyl or        Phenyl C₁₋₅₀ alkyl.

One embodiment of the present invention consists of a vaccineformulation comprising a polyoxyethylene ether of general formula (I),wherein n is between 1 and 50, preferably 4-24, most preferably 9; the Rcomponent is C₁₋₅₀, preferably C₄-C₂₀ alkyl and most preferably C₁₂alkyl, and A is a bond. The concentration of the polyoxyethylene ethersshould be in the range 0.1-20%, preferably from 0.1-10%, and mostpreferably in the range 0.1-1%. Preferred polyoxyethylene ethers areselected from the following group: polyoxyethylene-9-lauryl ether,polyoxyethylene-9-steoryl ether, polyoxyethylene-8-steoryl ether,polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether, andpolyoxyethylene-23-lauryl ether. Polyoxyethylene ethers such aspolyoxyethylene lauryl ether are described in the Merck index (12^(th)edition: entry 7717). These adjuvant molecules are described in WO99/52549.

The polyoxyethylene ether according to the general formula (I) abovemay, if desired, be combined with another adjuvant. For example, apreferred adjuvant combination is preferably with CpG as described inthe pending UK patent application GB 9820956.2.

Preferably a carrier is also present in the vaccine compositionaccording to the invention. The carrier may be an oil in water emulsion,or an aluminium salt, such as aluminium phosphate or aluminiumhydroxide.

A preferred oil-in-water emulsion comprises a metabolisible oil, such assqualene, alpha tocopherol and Tween 80. In a particularly preferredaspect the antigens in the vaccine composition according to theinvention are combined with QS21 and 3D-MPL in such an emulsion.Additionally the oil in water emulsion may contain span 85 and/orlecithin and/or tricaprylin.

Typically for human administration QS21 and 3D-MPL will be present in avaccine in the range of 1 μg-200 μg, such as 10-100 μg, preferably 10μg-50 μg per dose. Typically the oil in water will comprise from 2 to10% squalene, from 2 to 10% alpha tocopherol and from 0.3 to 3% tween80. Preferably the ratio of squalene: alpha tocopherol is equal to orless than 1 as this provides a more stable emulsion. Span 85 may also bepresent at a level of 1%. In some cases it may be advantageous that thevaccines of the present invention will further contain a stabiliser.

Non-toxic oil in water emulsions preferably contain a non-toxic oil,e.g. squalane or squalene, an emulsifier, e.g. Tween 80, in an aqueouscarrier. The aqueous carrier may be, for example, phosphate bufferedsaline.

A particularly potent adjuvant formulation involving QS21, 3D-MPL andtocopherol in an oil in water emulsion is described in WO 95/17210.

The present invention also provides a polyvalent vaccine compositioncomprising a vaccine formulation of the invention in combination withother antigens, in particular antigens useful for treating cancers, moreparticularly colorectal cancer, autoimmune diseases and relatedconditions. Such a polyvalent vaccine composition may include a TH-1inducing adjuvant as hereinbefore described.

According to another embodiment of this invention, an immunogeniccomposition described herein is delivered to a host via antigenpresenting cells (APCs), such as dendritic cells, macrophages, B cells,monocytes and other cells that may be engineered to be efficient APCs.Such cells may, but need not, be genetically modified to increase thecapacity for presenting the antigen, to improve activation and/ormaintenance of the T cell response, to have anti-tumor effects per seand/or to be immunologically compatible with the receiver (i.e., matchedHLA haplotype). APCs may generally be isolated from any of a variety ofbiological fluids and organs, including tumor and peritumoral tissues,and may be autologous, allogeneic, syngeneic or xenogeneic cells.

Certain preferred embodiments of the present invention use dendriticcells or progenitors thereof as antigen-presenting cells. Dendriticcells are highly potent APCs (Banchereau and Steinman, Nature392:245-251, 1998) and have been shown to be effective as aphysiological adjuvant for eliciting prophylactic or therapeuticantitumor immunity (see Timmerman and Levy, Ann. Rev. Med. 50:507-529,1999). In general, dendritic cells may be identified based on theirtypical shape (stellate in situ, with marked cytoplasmic processes(dendrites) visible in vitro), their ability to take up, process andpresent antigens with high efficiency and their ability to activatenaïve T cell responses. Dendritic cells may, of course, be engineered toexpress specific cell-surface receptors or ligands that are not commonlyfound on dendritic cells in vivo or ex vivo, and such modified dendriticcells are contemplated by the present invention. As an alternative todendritic cells, secreted vesicles antigen-loaded dendritic cells(called exosomes) may be used within a vaccine (see Zitvogel et al.,Nature Med. 4:594-600, 1998).

Dendritic cells and progenitors may be obtained from peripheral blood,bone marrow, tumor-infiltrating cells, peritumoral tissues-infiltratingcells, lymph nodes, spleen, skin, umbilical cord blood or any othersuitable tissue or fluid. For example, dendritic cells may bedifferentiated ex vivo by adding a combination of cytokines such asGM-CSF, IL-4, IL-13 and/or TNFα to cultures of monocytes harvested fromperipheral blood. Alternatively, CD34 positive cells harvested fromperipheral blood, umbilical cord blood or bone marrow may bedifferentiated into dendritic cells by adding to the culture mediumcombinations of GM-CSF, IL-3, TNFα, CD40 ligand, LPS, flt3 ligand and/orother compound(s) that induce differentiation, maturation andproliferation of dendritic cells.

Dendritic cells are conveniently categorized as “immature” and “mature”cells, which allows a simple way to discriminate between two wellcharacterized phenotypes. However, this nomenclature should not beconstrued to exclude all possible intermediate stages of differentiationImmature dendritic cells are characterized as APC with a high capacityfor antigen uptake and processing, which correlates with the highexpression of Fcγ receptor and mannose receptor. The mature phenotype istypically characterized by a lower expression of these markers, but ahigh expression of cell surface molecules responsible for T cellactivation such as class I and class II MHC, adhesion molecules (e.g.,CD54 and CD11) and costimulatory molecules (e.g., CD40, CD80, CD86 and4-1BB).

APCs may generally be transfected with a polynucleotide of the invention(or portion or other variant thereof) such that the encoded polypeptide,or an immunogenic portion thereof, is expressed on the cell surface.Such transfection may take place ex vivo, and a pharmaceuticalcomposition comprising such transfected cells may then be used fortherapeutic purposes, as described herein. Alternatively, a genedelivery vehicle that targets a dendritic or other antigen presentingcell may be administered to a patient, resulting in transfection thatoccurs in vivo. In vivo and ex vivo transfection of dendritic cells, forexample, may generally be performed using any methods known in the art,such as those described in WO 97/24447, or the gene gun approachdescribed by Mahvi et al., Immunology and cell Biology 75:456-460, 1997.Antigen loading of dendritic cells may be achieved by incubatingdendritic cells or progenitor cells with the tumor polypeptide, DNA(naked or within a plasmid vector) or RNA; or with antigen-expressingrecombinant bacterium or viruses (e.g., vaccinia, fowlpox, adenovirus orlentivirus vectors). Prior to loading, the polypeptide may be covalentlyconjugated to an immunological partner that provides T cell help (e.g.,a carrier molecule). Alternatively, a dendritic cell may be pulsed witha non-conjugated immunological partner, separately or in the presence ofthe polypeptide.

While any suitable carrier known to those of ordinary skill in the artmay be employed in the pharmaceutical compositions of this invention,the type of carrier will typically vary depending on the mode ofadministration. Compositions of the present invention may be formulatedfor any appropriate manner of administration, including for example,topical, oral, nasal, mucosal, intravenous, intracranial,intraperitoneal, subcutaneous and intramuscular administration.

Carriers for use within such pharmaceutical compositions arebiocompatible, and may also be biodegradable. In certain embodiments,the formulation preferably provides a relatively constant level ofactive component release. In other embodiments, however, a more rapidrate of release immediately upon administration may be desired. Theformulation of such compositions is well within the level of ordinaryskill in the art using known techniques. Illustrative carriers useful inthis regard include microparticles of poly(lactide-co-glycolide),polyacrylate, latex, starch, cellulose, dextran and the like. Otherillustrative delayed-release carriers include supramolecular biovectors,which comprise a non-liquid hydrophilic core (e.g., a cross-linkedpolysaccharide or oligosaccharide) and, optionally, an external layercomprising an amphiphilic compound, such as a phospholipid (see e.g.,U.S. Pat. No. 5,151,254 and PCT applications WO 94/20078, WO/94/23701and WO 96/06638). The amount of active compound contained within asustained release formulation depends upon the site of implantation, therate and expected duration of release and the nature of the condition tobe treated or prevented.

In another illustrative embodiment, biodegradable microspheres (e.g.,polylactate polyglycolate) are employed as carriers for the compositionsof this invention. Suitable biodegradable microspheres are disclosed,for example, in U.S. Pat. Nos. 4,897,268; 5,075,109; 5,928,647;5,811,128; 5,820,883; 5,853,763; 5,814,344, 5,407,609 and 5,942,252.Modified hepatitis B core protein carrier systems such as described inWO/99 40934, and references cited therein, will also be useful for manyapplications. Another illustrative carrier/delivery system employs acarrier comprising particulate-protein complexes, such as thosedescribed in U.S. Pat. No. 5,928,647, which are capable of inducing aclass I-restricted cytotoxic T lymphocyte responses in a host.

The pharmaceutical compositions of the invention will often furthercomprise one or more buffers (e.g., neutral buffered saline or phosphatebuffered saline), carbohydrates (e.g., glucose, mannose, sucrose ordextrans), mannitol, proteins, polypeptides or amino acids such asglycine, antioxidants, bacteriostats, chelating agents such as EDTA orglutathione, adjuvants (e.g., aluminum hydroxide), solutes that renderthe formulation isotonic, hypotonic or weakly hypertonic with the bloodof a recipient, suspending agents, thickening agents and/orpreservatives. Alternatively, compositions of the present invention maybe formulated as a lyophilizate.

The pharmaceutical compositions described herein may be presented inunit-dose or multi-dose containers, such as sealed ampoules or vials.Such containers are typically sealed in such a way to preserve thesterility and stability of the formulation until use. In general,formulations may be stored as suspensions, solutions or emulsions inoily or aqueous vehicles. Alternatively, a pharmaceutical compositionmay be stored in a freeze-dried condition requiring only the addition ofa sterile liquid carrier immediately prior to use.

The development of suitable dosing and treatment regimens for using theparticular compositions described herein in a variety of treatmentregimens, including e.g., oral, parenteral, intravenous, intranasal, andintramuscular administration and formulation, is well known in the art,some of which are briefly discussed below for general purposes ofillustration.

In certain applications, the pharmaceutical compositions disclosedherein may be delivered via oral administration to an animal. As such,these compositions may be formulated with an inert diluent or with anassimilable edible carrier, or they may be enclosed in hard- orsoft-shell gelatin capsule, or they may be compressed into tablets, orthey may be incorporated directly with the food of the diet.

The active compounds may even be incorporated with excipients and usedin the form of ingestible tablets, buccal tables, troches, capsules,elixirs, suspensions, syrups, wafers, and the like (see, for example,Mathiowitz et al., Nature 1997 Mar. 27; 386(6623):410-4; Hwang et al.,Crit Rev Ther Drug Carrier Syst 1998; 15(3):243-84; U.S. Pat. No.5,641,515; U.S. Pat. No. 5,580,579 and U.S. Pat. No. 5,792,451).Tablets, troches, pills, capsules and the like may also contain any of avariety of additional components, for example, a binder, such as gumtragacanth, acacia, cornstarch, or gelatin; excipients, such asdicalcium phosphate; a disintegrating agent, such as corn starch, potatostarch, alginic acid and the like; a lubricant, such as magnesiumstearate; and a sweetening agent, such as sucrose, lactose or saccharinmay be added or a flavoring agent, such as peppermint, oil ofwintergreen, or cherry flavoring. When the dosage unit form is acapsule, it may contain, in addition to materials of the above type, aliquid carrier. Various other materials may be present as coatings or tootherwise modify the physical form of the dosage unit. For instance,tablets, pills, or capsules may be coated with shellac, sugar, or both.Of course, any material used in preparing any dosage unit form should bepharmaceutically pure and substantially non-toxic in the amountsemployed. In addition, the active compounds may be incorporated intosustained-release preparation and formulations.

Typically, these formulations will contain at least about 0.1% of theactive compound or more, although the percentage of the activeingredient(s) may, of course, be varied and may conveniently be betweenabout 1 or 2% and about 60% or 70% or more of the weight or volume ofthe total formulation. Naturally, the amount of active compound(s) ineach therapeutically useful composition may be prepared is such a waythat a suitable dosage will be obtained in any given unit dose of thecompound. Factors such as solubility, bioavailability, biologicalhalf-life, route of administration, product shelf life, as well as otherpharmacological considerations will be contemplated by one skilled inthe art of preparing such pharmaceutical formulations, and as such, avariety of dosages and treatment regimens may be desirable.

For oral administration the compositions of the present invention mayalternatively be incorporated with one or more excipients in the form ofa mouthwash, dentifrice, buccal tablet, oral spray, or sublingualorally-administered formulation. Alternatively, the active ingredientmay be incorporated into an oral solution such as one containing sodiumborate, glycerin and potassium bicarbonate, or dispersed in adentifrice, or added in a therapeutically-effective amount to acomposition that may include water, binders, abrasives, flavoringagents, foaming agents, and humectants. Alternatively the compositionsmay be fashioned into a tablet or solution form that may be placed underthe tongue or otherwise dissolved in the mouth.

In certain circumstances it will be desirable to deliver thepharmaceutical compositions disclosed herein parenterally,intravenously, intramuscularly, or even intraperitoneally. Suchapproaches are well known to the skilled artisan, some of which arefurther described, for example, in U.S. Pat. No. 5,543,158; U.S. Pat.No. 5,641,515 and U.S. Pat. No. 5,399,363. In certain embodiments,solutions of the active compounds as free base or pharmacologicallyacceptable salts may be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions may also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations generally will contain a preservative to prevent the growthof microorganisms.

Illustrative pharmaceutical forms suitable for injectable use includesterile aqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions (for example, see U.S. Pat. No. 5,466,468). In all cases theform must be sterile and must be fluid to the extent that easysyringability exists. It must be stable under the conditions ofmanufacture and storage and must be preserved against the contaminatingaction of microorganisms, such as bacteria and fungi. The carrier can bea solvent or dispersion medium containing, for example, water, ethanol,polyol (e.g., glycerol, propylene glycol, and liquid polyethyleneglycol, and the like), suitable mixtures thereof, and/or vegetable oils.Proper fluidity may be maintained, for example, by the use of a coating,such as lecithin, by the maintenance of the required particle size inthe case of dispersion and/or by the use of surfactants. The preventionof the action of microorganisms can be facilitated by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars or sodium chloride. Prolonged absorption of the injectablecompositions can be brought about by the use in the compositions ofagents delaying absorption, for example, aluminum monostearate andgelatin.

In one embodiment, for parenteral administration in an aqueous solution,the solution should be suitably buffered if necessary and the liquiddiluent first rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, a sterile aqueous medium that can be employed will be knownto those of skill in the art in light of the present disclosure. Forexample, one dosage may be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion, (see for example, “Remington's PharmaceuticalSciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variationin dosage will necessarily occur depending on the condition of thesubject being treated. Moreover, for human administration, preparationswill of course preferably meet sterility, pyrogenicity, and the generalsafety and purity standards as required by FDA Office of Biologicsstandards.

In another embodiment of the invention, the compositions disclosedherein may be formulated in a neutral or salt form. Illustrativepharmaceutically-acceptable salts include the acid addition salts(formed with the free amino groups of the protein) and which are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, oxalic, tartaric, mandelic, andthe like. Salts formed with the free carboxyl groups can also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, histidine, procaine and the like. Upon formulation,solutions will be administered in a manner compatible with the dosageformulation and in such amount as is therapeutically effective.

The carriers can further comprise any and all solvents, dispersionmedia, vehicles, coatings, diluents, antibacterial and antifungalagents, isotonic and absorption delaying agents, buffers, carriersolutions, suspensions, colloids, and the like. The use of such mediaand agents for pharmaceutical active substances is well known in theart. Except insofar as any conventional media or agent is incompatiblewith the active ingredient, its use in the therapeutic compositions iscontemplated. Supplementary active ingredients can also be incorporatedinto the compositions. The phrase “pharmaceutically-acceptable” refersto molecular entities and compositions that do not produce an allergicor similar untoward reaction when administered to a human.

In certain embodiments, the pharmaceutical compositions may be deliveredby intranasal sprays, inhalation, and/or other aerosol deliveryvehicles. Methods for delivering genes, nucleic acids, and peptidecompositions directly to the lungs via nasal aerosol sprays has beendescribed, e.g., in U.S. Pat. No. 5,756,353 and U.S. Pat. No. 5,804,212.Likewise, the delivery of drugs using intranasal microparticle resins(Takenaga et al., J Controlled Release 1998 Mar. 2; 52(1-2):81-7) andlysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725,871) are alsowell-known in the pharmaceutical arts. Likewise, illustrativetransmucosal drug delivery in the form of a polytetrafluoroetheylenesupport matrix is described in U.S. Pat. No. 5,780,045.

In certain embodiments, liposomes, nanocapsules, microparticles, lipidparticles, vesicles, and the like, are used for the introduction of thecompositions of the present invention into suitable hostcells/organisms. In particular, the compositions of the presentinvention may be formulated for delivery either encapsulated in a lipidparticle, a liposome, a vesicle, a nanosphere, or a nanoparticle or thelike. Alternatively, compositions of the present invention can be bound,either covalently or non-covalently, to the surface of such carriervehicles.

The formation and use of liposome and liposome-like preparations aspotential drug carriers is generally known to those of skill in the art(see for example, Lasic, Trends Biotechnol 1998 July; 16(7):307-21;Takakura, Nippon Rinsho 1998 March; 56(3):691-5; Chandran et al., IndianJ Exp Biol. 1997 August; 35(8):801-9; Margalit, Crit Rev Ther DrugCarrier Syst. 1995; 12(2-3):233-61; U.S. Pat. No. 5,567,434; U.S. Pat.No. 5,552,157; U.S. Pat. No. 5,565,213; U.S. Pat. No. 5,738,868 and U.S.Pat. No. 5,795,587, each specifically incorporated herein by referencein its entirety).

Liposomes have been used successfully with a number of cell types thatare normally difficult to transfect by other procedures, including Tcell suspensions, primary hepatocyte cultures and PC 12 cells (Renneisenet al., J Biol Chem. 1990 Sep. 25; 265(27):16337-42; Muller et al., DNACell Biol. 1990 April; 9(3):221-9). In addition, liposomes are free ofthe DNA length constraints that are typical of viral-based deliverysystems. Liposomes have been used effectively to introduce genes,various drugs, radiotherapeutic agents, enzymes, viruses, transcriptionfactors, allosteric effectors and the like, into a variety of culturedcell lines and animals. Furthermore, he use of liposomes does not appearto be associated with autoimmune responses or unacceptable toxicityafter systemic delivery.

In certain embodiments, liposomes are formed from phospholipids that aredispersed in an aqueous medium and spontaneously form multilamellarconcentric bilayer vesicles (also termed multilamellar vesicles (MLVs).

Alternatively, in other embodiments, the invention provides forpharmaceutically-acceptable nanocapsule formulations of the compositionsof the present invention. Nanocapsules can generally entrap compounds ina stable and reproducible way (see, for example, Quintanar-Guerrero etal., Drug Dev Ind Pharm. 1998 December; 24(12):1113-28). To avoid sideeffects due to intracellular polymeric overloading, such ultrafineparticles (sized around 0.1 μm) may be designed using polymers able tobe degraded in vivo. Such particles can be made as described, forexample, by Couvreur et al., Crit Rev Ther Drug Carrier Syst. 1988;5(1):1-20; zur Muhlen et al., Eur J Pharm Biopharm. 1998 March;45(2):149-55; Zambaux et al. J Controlled Release. 1998 Jan. 2;50(1-3):31-40; and U.S. Pat. No. 5,145,684.

This invention also relates to the use of polynucleotides, in the formof primers derived from the polynucleotides of the present invention,and of polypeptides, in the form of antibodies or reagents specific forthe polypeptide of the present invention, as diagnostic reagents.

The identification of genetic or biochemical markers in blood or tissuesthat will enable the detection of very early changes along thecarcinogenesis pathway will help in determining the best treatment forthe patient. Surrogate tumour markers, such as polynucleotideexpression, can be used to diagnose different forms and states ofcancer. The identification of expression levels of the polynucleotidesof the invention will be useful in both the staging of the cancerousdisorder and grading the nature of the cancerous tissue. The stagingprocess monitors the advancement of the cancer and is determined on thepresence or absence of malignant tissue in the areas biopsied. Thepolynucleotides of the invention can help to perfect the staging processby identifying markers for the aggresivity of a cancer, for example thepresence in different areas of the body. The grading of the cancerdescribes how closely a tumour resembles normal tissue of its same typeand is assessed by its cell morphology and other markers ofdifferentiation. The polynucleotides of the invention can be useful indetermining the tumour grade as they can help in the determination ofthe differentiation status of the cells of a tumour.

The diagnostic assays offer a process for diagnosing or determining asusceptibility to cancers, autoimmune disease and related conditionsthrough diagnosis by methods comprising determining from a samplederived from a subject an abnormally decreased or increased level ofpolypeptide or mRNA. This method of diagnosis is known as differentialexpression. The expression of a particular gene is compared between adiseased tissue and a normal tissue. A difference between thepolynucleotide-related gene, mRNA, or protein in the two tissues iscompared, for example in molecular weight, amino acid or nucleotidesequence, or relative abundance, indicates a change in the gene, or agene which regulates it, in the tissue of the human that was suspectedof being diseased.

Decreased or increased expression can be measured at the RNA level.PolyA RNA is first isolated from the two tissues and the detection ofmRNA encoded by a gene corresponding to a differentially expressedpolynucleotide of the invention can be detected by, for example, in situhybridization in tissue sections, reverse trascriptase-PCR, usingNorthern blots containing poly A+ mRNA, or any other direct or inderectRNA detection method. An increased or decreased expression of a givenRNA in a diseased tissue compared to a normal tissue suggests that thetranscript and/or the expressed protein has a role in the disease. Thusdetection of a higher or lower level of mRNA corresponding to SEQ ID NO:1 relative to normal level is indicative of the presence of cancer inthe patient.

mRNA expression levels in a sample can be determined by generation of alibrary of expressed sequence tags (ESTs) from the sample. The relativerepresentation of ESTs in the library can be used to assess the relativerepresentation of the gene transcript in the starting sample. The ESTanalysis of the test can then be compared to the EST analysis of areference sample to determine the relative expression levels of thepolynucleotide of interest.

Other mRNA analyses can be carried out using serial analysis of geneexpression (SAGE) methodology (Velculescu et. Al. Science (1995)270:484), differential display methodology (For example, U.S. Pat. No.5,776,683) or hybridization analysis which relies on the specificity ofnucleotide interactions.

Alternatively, the comparison could be made at the protein level. Theprotein sizes in the two tissues may be compared using antibodies todetect polypeptides in Western blots of protein extracts from the twotissues. Expression levels and subcellular localization may also bedetected immunologically using antibodies to the corresponding protein.Further assay techniques that can be used to determine levels of aprotein, such as a polypeptide of the present invention, in a samplederived from a host are well-known to those of skill in the art. Araised or decreased level of polypeptide expression in the diseasedtissue compared with the same protein expression level in the normaltissue indicates that the expressed protein may be involved in thedisease.

In the assays of the present invention, the diagnosis can be determinedby detection of gene product expression levels encoded by at least onesequence set forth in SEQ ID NO: 1. A comparison of the mRNA or proteinlevels in a diseased versus normal tissue may also be used to follow theprogression or remission of a disease.

A large number of polynucleotide sequences in a sample can be assayedusing polynucleotide arrays. These can be used to examine differentialexpression of genes and to determine gene function. For example, arraysof the polynucleotide sequences SEQ ID NO:1 can be used to determine ifany of the polynucleotides are differentially expressed between a normaland cancer cell. In one embodiment of the invention, an array ofoligonucleotides probes comprising the SEQ ID NO:1 nucleotide sequenceor fragments thereof can be constructed to conduct efficient screeningof e.g., genetic mutations. Array technology methods are well known andhave general applicability and can be used to address a variety ofquestions in molecular genetics including gene expression, geneticlinkage, and genetic variability (see for example: M. Chee et al.,Science, Vol 274, pp 610-613 (1996)).

“Diagnosis” as used herein includes determination of a subject'ssusceptibility to a disease, determination as to whether a subjectpresently has the disease, and also the prognosis of a subject affectedby the disease.

The present invention, further relates to a diagnostic kit forperforming a diagnostic assay which comprises:

(a) a polynucleotide of the present invention, preferably the nucleotidesequence of SEQ ID NO: 1, or a fragment thereof;

(b) a nucleotide sequence complementary to that of (a), preferably thenucleotide sequence of SEQ ID NO: 6;

(c) a polypeptide of the present invention, preferably the polypeptideof SEQ ID NO: 2 or 3, or a fragment thereof; or

(d) an antibody to a polypeptide of the present invention, preferably tothe polypeptide of SEQ ID NO:2 or 3.

The nucleotide sequences of the present invention are also valuable forchromosomal localisation. The sequence is specifically targeted to, andcan hybridize with, a particular location on an individual humanchromosome. The mapping of relevant sequences to chromosomes accordingto the present invention is an important first step in correlating thosesequences with gene associated disease. Once a sequence has been mappedto a precise chromosomal location, the physical position of the sequenceon the chromosome can be correlated with genetic map data. Such data arefound in, for example, V. McKusick, Mendelian Inheritance in Man(available on-line through Johns Hopkins University Welch MedicalLibrary). The relationship between genes and diseases that have beenmapped to the same chromosomal region are then identified throughlinkage analysis (coinheritance of physically adjacent genes).Thedifferences in the cDNA or genomic sequence between affected andunaffected individuals can also be determined.

The polypeptides of the invention or their fragments or analogs thereof,or cells expressing them, can also be used as immunogens to produceantibodies immunospecific for polypeptides of the present invention. Theterm “immunospecific” means that the antibodies have substantiallygreater affinity for the polypeptides of the invention than theiraffinity for other related polypeptides in the prior art.

In a further aspect the invention provides an antibody immunospecificfor a polypeptide according to the invention or an immunologicalfragment thereof as hereinbefore defined. Preferably the antibody is amonoclonal antibody.

Antibodies generated against polypeptides of the present invention maybe obtained by administering the polypeptides or epitope-bearingfragments, analogs or cells to an animal, preferably a non-human animal,using routine protocols. For preparation of monoclonal antibodies, anytechnique which provides antibodies produced by continuous cell linecultures can be used. Examples include the hybridoma technique (Kohler,G. and Milstein, C., Nature (1975) 256:495-497), the trioma technique,the human B-cell hybridoma technique (Kozbor et al., Immunology Today(1983) 4:72) and the EBV-hybridoma technique (Cole et al., MonoclonalAntibodies and Cancer Therapy, 77-96, Alan R. Liss, Inc., 1985).

Techniques for the production of single chain antibodies, such as thosedescribed in U.S. Pat. No. 4,946,778, can also be adapted to producesingle chain antibodies to polypeptides of this invention. Also,transgenic mice, or other organisms, including other mammals, may beused to express humanized antibodies.

The above-described antibodies may be employed to isolate or to identifyclones expressing the polypeptide or to purify the polypeptides byaffinity chromatography.

The antibody of the invention may also be employed to prevent or treatcancer, particularly colorectal cancer, autoimmune disease and relatedconditions.

Another aspect of the invention relates to a method for inducing ormodulating an immunological response in a mammal which comprisesinoculating the mammal with a polypeptide of the present invention,adequate to produce antibody and/or T cell immune response to protect orameliorate the symptoms or progression of the disease. Yet anotheraspect of the invention relates to a method of inducing or modulatingimmunological response in a mammal which comprises, delivering apolypeptide of the present invention via a vector directing expressionof the polynucleotide and coding for the polypeptide in vivo in order toinduce such an immunological response to produce antibody to protectsaid animal from diseases.

It will be appreciated that the present invention therefore provides amethod of treating abnormal conditions such as, for instance, cancer andautoimmune diseases, in particular, colorectal cancer, related to eithera presence of, an excess of, or an under-expression of, CASB7439polypeptide activity. Other abnormal conditions related to CASB7439expression that the invention seeks to treat are chronic lymphocyticleukemiae and germ cell tumours.

The present invention further provides for a method of screeningcompounds to identify those which stimulate or which inhibit thefunction of the CASB7439 polypeptide. In general, agonists orantagonists may be employed for therapeutic and prophylactic purposesfor such diseases as hereinbefore mentioned. Compounds may be identifiedfrom a variety of sources, for example, cells, cell-free preparations,chemical libraries, and natural product mixtures. Such agonists,antagonists or inhibitors so-identified may be natural or modifiedsubstrates, ligands, receptors, enzymes, etc., as the case may be, ofthe polypeptide; or may be structural or functional mimetics thereof(see Coligan et al., Current Protocols in Immunology 1(2):Chapter 5(1991)). Screening methods will be known to those skilled in the art.Further screening methods may be found in for example D. Bennett et al.,J Mol Recognition, 8:52-58 (1995); and K. Johanson et al., J Biol Chem,270(16):9459-9471 (1995) and references therein.

Thus the invention provides a method for screening to identify compoundswhich stimulate or which inhibit the function of the polypeptide of theinvention which comprises a method selected from the group consistingof:

(a) measuring the binding of a candidate compound to the polypeptide (orto the cells or membranes bearing the polypeptide) or a fusion proteinthereof by means of a label directly or indirectly associated with thecandidate compound;

(b) measuring the binding of a candidate compound to the polypeptide (orto the cells or membranes bearing the polypeptide) or a fusion proteinthereof in the presense of a labeled competitior;

(c) testing whether the candidate compound results in a signal generatedby activation or inhibition of the polypeptide, using detection systemsappropriate to the cells or cell membranes bearing the polypeptide;

(d) mixing a candidate compound with a solution containing a polypeptideof claim 1, to form a mixture, measuring activity of the polypeptide inthe mixture, and comparing the activity of the mixture to a standard; or

(e) detecting the effect of a candidate compound on the production ofmRNA encoding said polypeptide and said polypeptide in cells, using forinstance, an ELISA assay.

The polypeptide of the invention may be used to identify membrane boundor soluble receptors, if any, through standard receptor bindingtechniques known in the art. Well known screening methods may also beused to identify agonists and antagonists of the polypeptide of theinvention which compete with the binding of the polypeptide of theinvention to its receptors, if any.

Thus, in another aspect, the present invention relates to a screeningkit for identifying agonists, antagonists, ligands, receptors,substrates, enzymes, etc. for polypeptides of the present invention; orcompounds which decrease or enhance the production of such topolypeptides, which comprises:

(a) a polypeptide of the present invention;

(b) a recombinant cell expressing a polypeptide of the presentinvention;

(c) a cell membrane expressing a polypeptide of the present invention;or

(d) antibody to a polypeptide of the present invention;

which polypeptide is preferably that of SEQ ID NO:2 or 3.

It will be readily appreciated by the skilled artisan that a polypeptideof the present invention may also be used in a method for thestructure-based design of an agonist, antagonist or inhibitor of thepolypeptide, by:

(a) determining in the first instance the three-dimensional structure ofthe polypeptide;

(b) deducing the three-dimensional structure for the likely reactive orbinding site(s) of an agonist, antagonist or inhibitor;

(c) synthesing candidate compounds that are predicted to bind to orreact with the deduced binding or reactive site; and

(d) testing whether the candidate compounds are indeed agonists,antagonists or inhibitors.

Gene therapy may also be employed to effect the endogenous production ofCASB7439 polypeptide by the relevant cells in the subject. For anoverview of gene therapy, see Chapter 20, Gene Therapy and otherMolecular Genetic-based Therapeutic Approaches, (and references citedtherein) in Human Molecular Genetics, T Strachan and A P Read, BIOSScientific Publishers Ltd (1996).

Vaccine preparation is generally described in PharmaceuticalBiotechnology, Vol. 61 Vaccine Design—the subunit and adjuvant approach,edited by Powell and Newman, Plenum Press, 1995. New Trends andDevelopments in Vaccines, edited by Voller et al., University ParkPress, Baltimore, Md., U.S.A. 1978. Encapsulation within liposomes isdescribed, for example, by Fullerton, U.S. Pat. No. 4,235,877.Conjugation of proteins to macromolecules is disclosed, for example, byLikhite, U.S. Pat. No. 4,372,945 and by Armor et al., U.S. Pat. No.4,474,757.

The amount of protein in each vaccine dose is selected as an amountwhich induces an immunoprotective response without significant, adverseside effects in typical vaccinees. Such amount will vary depending uponwhich specific immunogen is employed. Generally, it is expected thateach dose will comprise 1-1000 μg of protein, preferably 2-100 μg, mostpreferably 4-40 μg. An optimal amount for a particular vaccine can beascertained by standard studies involving observation of antibody titresand other responses in subjects. Following an initial vaccination,subjects may receive a boost in about 4 weeks.

“Isolated” means altered “by the hand of man” from the natural state. Ifan “isolated” composition or substance occurs in nature, it has beenchanged or removed from its original environment, or both. For example,a polynucleotide or a polypeptide naturally present in a living animalis not “isolated,” but the same polynucleotide or polypeptide separatedfrom the coexisting materials of its natural state is “isolated”, as theterm is employed herein.

“Polynucleotide” generally refers to any polyribonucleotide orpolydeoxribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA including single and double stranded regions.

“Variant” refers to a polynucleotide or polypeptide that differs from areference polynucleotide or polypeptide, but retains essentialproperties. A typical variant of a polynucleotide differs in nucleotidesequence from another, reference polynucleotide. Changes in thenucleotide sequence of the variant may or may not alter the amino acidsequence of a polypeptide encoded by the reference polynucleotide.Nucleotide changes may result in amino acid substitutions, additions,deletions, fusions and truncations in the polypeptide encoded by thereference sequence, as discussed below. A typical variant of apolypeptide differs in amino acid sequence from another, referencepolypeptide. Generally, differences are limited so that the sequences ofthe reference polypeptide and the variant are closely similar overalland, in many regions, identical. A variant and reference polypeptide maydiffer in amino acid sequence by one or more substitutions, additions,deletions in any combination. A substituted or inserted amino acidresidue may or may not be one encoded by the genetic code. A variant ofa polynucleotide or polypeptide may be a naturally occurring such as anallelic variant, or it may be a variant that is not known to occurnaturally. Non-naturally occurring variants of polynucleotides andpolypeptides may be made by mutagenesis techniques or by directsynthesis.

“Identity,” as known in the art, is a relationship between two or morepolypeptide sequences or two or more polynucleotide sequences, asdetermined by comparing the sequences. In the art, “identity” also meansthe degree of sequence relatedness between polypeptide or polynucleotidesequences, as the case may be, as determined by the match betweenstrings of such sequences. “Identity” and “similarity” can be readilycalculated by known methods, including but not limited to thosedescribed in (Computational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; Biocomputing: Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; and Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York,1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073(1988). Preferred methods to determine identity are designed to give thelargest match between the sequences tested. Methods to determineidentity and similarity are codified in publicly available computerprograms. Preferred computer program methods to determine identity andsimilarity between two sequences include, but are not limited to, theGCG program package (Devereux, J., et al., Nucleic Acids Research 12(1):387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S. F. et al., J. Molec.Biol. 215: 403-410 (1990). The BLAST X program is publicly availablefrom NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBINLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol. 215:403-410 (1990). The well known Smith Waterman algorithm may also be usedto determine identity.

The preferred algorithm used is FASTA. The preferred parameters forpolypeptide or polynuleotide sequence comparison using this algorithminclude the following:

Gap Penalty: 12

Gap extension penalty: 4

Word size: 2, max 6

Preferred parameters for polypeptide sequence comparison with othermethods include the following:

1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970)

Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl.Acad. Sci. USA. 89:10915-10919 (1992)

Gap Penalty: 12

Gap Length Penalty: 4

A program useful with these parameters is publicly available as the“gap” program from Genetics Computer Group, Madison Wis. Theaforementioned parameters are the default parameters for polypeptidecomparisons (along with no penalty for end gaps).

Preferred parameters for polynucleotide comparison include thefollowing:

1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970)

Comparison matrix: matches=+10, mismatch=0

Gap Penalty: 50

Gap Length Penalty: 3

A program useful with these parameters is publicly available as the“gap” program from Genetics Computer Group, Madison Wis. Theaforementioned parameters are the default parameters for polynucleotidecomparisons.

By way of example, a polynucleotide sequence of the present inventionmay be identical to the reference sequence of SEQ ID NO:1, that is be100% identical, or it may include up to a certain integer number ofnucleotide alterations as compared to the reference sequence. Suchalterations are selected from the group consisting of at least onenucleotide deletion, substitution, including transition andtransversion, or insertion, and wherein said alterations may occur atthe 5′ or 3′ terminal positions of the reference nucleotide sequence oranywhere between those terminal positions, interspersed eitherindividually among the nucleotides in the reference sequence or in oneor more contiguous groups within the reference sequence. The number ofnucleotide alterations is determined by multiplying the total number ofnucleotides in SEQ ID NO:1 by the numerical percent of the respectivepercent identity(divided by 100) and subtracting that product from saidtotal number of nucleotides in SEQ ID NO:1, or:

n _(n) ≦x _(n)−(x _(n) ·y),

wherein n_(n) is the number of nucleotide alterations, x_(n) is thetotal number of nucleotides in SEQ ID NO:1, and y is, for instance, 0.70for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for 95%,etc.,and wherein any non-integer product of x_(n) and y is rounded down tothe nearest integer prior to subtracting it from x_(n). Alterations of apolynucleotide sequence encoding the polypeptide of SEQ ID NO:2 maycreate nonsense, missense or frameshift mutations in this codingsequence and thereby alter the polypeptide encoded by the polynucleotidefollowing such alterations.

Similarly, a polypeptide sequence of the present invention may beidentical to the reference sequence of SEQ ID NO:2, that is be 100%identical, or it may include up to a certain integer number of aminoacid alterations as compared to the reference sequence such that the %identity is less than 100%. Such alterations are selected from the groupconsisting of at least one amino acid deletion, substitution, includingconservative and non-conservative substitution, or insertion, andwherein said alterations may occur at the amino- or carboxy-terminalpositions of the reference polypeptide sequence or anywhere betweenthose terminal positions, interspersed either individually among theamino acids in the reference sequence or in one or more contiguousgroups within the reference sequence. The number of amino acidalterations for a given % identity is determined by multiplying thetotal number of amino acids in SEQ ID NO:2 by the numerical percent ofthe respective percent identity(divided by 100) and then subtractingthat product from said total number of amino acids in SEQ ID NO:2, or:

n _(a) ≦x _(a)−(x _(a) ·y),

wherein n_(a) is the number of amino acid alterations, x_(a) is thetotal number of amino acids in SEQ ID NO:2, and y is, for instance 0.70for 70%, 0.80 for 80%, 0.85 for 85% etc., and wherein any non-integerproduct of x_(a) and y is rounded down to the nearest integer prior tosubtracting it from x_(a).

“Homolog” is a generic term used in the art to indicate a polynucleotideor polypeptide sequence possessing a high degree of sequence relatednessto a subject sequence. Such relatedness may be quantified by determiningthe degree of identity and/or similarity between the sequences beingcompared as hereinbefore described. Falling within this generic term arethe terms “ortholog”, meaning a polynucleotide or polypeptide that isthe functional equivalent of a polynucleotide or polypeptide in anotherspecies and “paralog” meaning a functionally similar sequence whenconsidered within the same species.

Examples Example 1

Real-Time RT-PCR Analysis

Real-time RT-PCR (U. Gibson. 1996. Genome Research: 6,996) is used tocompare mRNA transcript abundance of the candidate antigen in matchedtumour and normal colon tissues from multiple patients. In addition,mRNA levels of the candidate gene in a panel of normal tissues are alsoevaluated by this approach.

Total RNA from normal and tumour colon is extracted from snap frozenbiopsies using TriPure reagent (Boehringer). Total RNA from normaltissues is purchased from InVitrogen or is extracted from snap frozenbiopsies using TriPure reagent. Poly-A+ mRNA is purified from total RNAafter DNAase treatment using oligo-dT magnetic beads (Dynal).Quantification of the mRNA is performed by spectrofluorimetry(VersaFluor, BioRad) using SybrII dye (Molecular Probes). Primers forreal-time PCR amplification are designed with the Perkin-Elmer PrimerExpress software using default options for TaqMan amplificationconditions.

Real-time reactions are assembled according to standard PCR protocolsusing 2 ng of purified mRNA for each reaction. SybrII dye (MolecularProbes) is added at a final dilution of 1/75000 for real-time detection.Amplification (40 cycles) and real-time detection is performed in aPerkin-Elmer Biosystems PE7700 system using conventional instrumentsettings. Ct values are calculated using the PE7700 Sequence Detectorsoftware. Several Ct values are obtained for each samples : for thepatient samples, the tumour Ct (CtT) and the matched normal colon Ct(CtN) values on the candidate TAA, and for the panel of normal tissuesamples, a CtXY for each normal tissue XY. An another Ct (CtA) is alsocalculated on Actin gene, as an internal reference, for all of thesamples. Alternatively, real-time PCR amplification can be monitoredusing a Taqman probe. Amplification (40 cycles) and real-time detectionis performed in a Perkin-Elmer Biosystems PE7700 system usingconventional instrument settings. Ct values are calculated using thePE7700 Sequence Detector Software. Ct values are obtained from eachtissue sample for the target mRNA (CtX) and for the actin mRNA (CtA).

As the efficiency of PCR amplification under the prevailing experimentalconditions is close to the theoretical amplification efficiency,2^((CtN/T/XY−CtA)) value is an estimate of the relative TAA transcriptlevel of the sample, standardised with respect to Actin transcriptlevel. A value of 1 thus suggests the candidate antigen and Actin havethe same expression level.

Real-time PCR reactions were first performed on tumour colon andmatching normal colon from biopsies of 12 patients. Reactions were thenperformed on a more complete data set totalling 18 patients (areincluded in this data set the first 12 patients). Duplicates for 6 outof these 18 patients were made in this data set. Six further patientswere tested, and the results were pooled with the previous 18. Thestatistics on the final pool are shown in table 3, and illustrated inFIG. 1.

A series of 48 normal tissue samples, representing 29 different tissues,were also tested by the same procedure (analysed normal tissues aregiven in table 3). TAA transcript levels are calculated as describedabove. The proportion of patients over-expressing the candidate antigen,as well as the average transcript over-expression versus normal tissuesis also calculated from this data set. The results are illustrated inFIG. 1.

TABLE 1 CASB7439 Real-time PCR expression results: data set of 12patients. % of patients with a mRNA level higher in matched tumour 92%colon (positive patients) % of patients with a mRNA level at least 3fold higher in 92% matched tumour colon % of patients with a mRNA levelat least 10 fold higher in 92% matched tumour colon % of patients with amRNA level at least 3 fold lower in  8% matched tumour colon. Averagematched normal colon mRNA level (Actin 0.0026 standardised) Averagematched tumour colon mRNA level in positive 0.265 patients (Actinstandardised) Average mRNA over-expression fold 2028 Median mRNAover-expression fold 115 Average normal tissues mRNA level 0.0079 Mediannormal tissues mRNA level 0.0016 Average normal tissues mRNA level0.0064 Median normal tissues mRNA level 0.0017 % of patients with a mRNAlevel higher than average normal 92% tissues % of patients with a mRNAlevel higher than 10 fold average 75% normal tissues Normalnon-dispensable tissues higher than median normal None tissue mRNA level

TABLE 2 CASB7439 Real-time PCR expression results: data set of 18patients. % of patients with a mRNA level higher in matched tumour 89%colon (positive patients) % of patients with a mRNA level at least 3fold higher in 89% matched tumour colon % of patients with a mRNA levelat least 10 fold higher in 78% matched tumour colon % of patients with amRNA level at least 3 fold lower in  5% matched tumour colon. Averagematched normal colon mRNA level (Actin 0.005 standardised) Averagematched tumour colon mRNA level in positive 0.152 patients (Actinstandardised) Average mRNA over-expression fold 1100 Median mRNAover-expression fold 60 Average normal tissues mRNA level 0.0065 Mediannormal tissues mRNA level 0.0015 Average normal tissues mRNA level 0.005Median normal tissues mRNA level 0.0015 % of patients with a mRNA levelhigher than median normal 94% tissues % of patients with a mRNA levelhigher than 10 fold median 94% normal tissues Normal non-dispensabletissues higher than median normal None tissue mRNA level

TABLE 3 CASB7439 Real-time PCR expression results: data set of 24patients % of patients with a CASB7439 transcript level higher in tumour92% colon than adjacent normal colon (positive patients) % of positivepatients with a CASB7439 transcript level at least 75% 10 fold higher intumour colon than adjacent normal colon Average transcriptover-expression fold in tumors of positive 1289 patients % of patientswith a CASB7439 transcript level higher in tumour 96% colon than normaltissue average % of patients with a mRNA level at least 10 fold higherin 62.5%  tumour colon than normal tissue average Normal tissues whereCASB7439 transcript expression is none equivalent to tumour transcriptlevel in tumours

Real-time PCR reactions were also performed using the Taqman protocol(as described above) on tumour colon and adjacent normal colon frombiopsies of 6 patients. Three replicate measures were taken for each,and the average was used for further calculations. Results are shown inFIG. 1. Moreover, 36 normal tissue samples, representing 28 differenttissues (see table 5), were also tested by the same procedure. Resultsare shown in FIG. 2.

TABLE 4 CASB7439 Real-time PCR expression results using Taqman probeNumber of tumor samples from different patients  6 % of patients with aCASB7439 transcript level higher in tumour 100% colon than adjacentnormal colon (positive patients) % of positive patients with a CASB7439transcript level at least 10  83% fold higher in tumour colon thanadjacent normal colon Average transcript over-expression fold in tumorsof positive 109 patients % of patients with a CASB7439 transcript levelhigher in tumour 100% colon than normal tissue average % of patientswith a mRNA level at least 10 fold higher in 100% tumour colon thannormal tissue average Normal tissues where CASB7439 transcriptexpression is none equivalent to tumour transcript level in tumours

The results clearly suggest CASB7439 transcript is over-expressed incolorectal tumours compared to adjacent normal colon and to all of theabove mentioned normal tissues. More than 90% of the patients stronglyover-express CASB7439 transcript in tumour, as compared to adjacentnormal colon. Average over-expression fold in the tumors is at least of100. Moreover, more than 90% of the patients over-express the CASB7439transcript in colorectal tumors as compared to other normal tissues,more than 60% of them over-expressing it at least 10 fold.

TABLE 5 listing of normal tissues used for CASB7439 transcriptexpression analysis. Tissue Abbreviation Adrenal gland Ad_Gl Aorta AoBladder Bl Bone marrow Bo_Ma Brain Bra Cervix Ce Colon Co Fallopian tubeFa_Tu Heart He Ileon Il Kidney Ki Liver Li Lung Lu Lymph node Ly_NoOesophagus Oe Parathyroid gland Pa_Thy Rectum Re Skin Sk Skeletal muscleSk_Mu Small intestine Sm_In Spleen Sp Stomach St Thyroid gland ThyTrachea Tra Ovary Ov Placenta Pl Prostate Pr Testis Te

Example 2

Differential Screening of cDNA Arrays.

Identification of tumour-associated genes in the subtracted cDNA libraryis accomplished by differential screening.

Total bacterial DNA is extracted from 100 μl over-night cultures.Bacteria are lysed with guanidium isothiocyantate and the bacterial DNAis affinity purified using magnetic glass (Boehringer). Plasmid insertsare recovered from the bacterial DNA by Advantage PCR amplification(Clontech). The PCR products are dotted onto two nylon membranes toproduce high density cDNA arrays using the Biomek 96 HDRT tool(Beekman). The spotted cDNA is covalently linked to the membrane by UVirradiation. The first membrane is hybridised with a mixed cDNA probeprepared from the tumour of a single patient. The second membrane ishybridised with an equivalent amount of mixed cDNA probe prepared fromnormal colon of the same patient. The probe cDNA is prepared by PCRamplification as described above and is labelled using the AlkPhosDirect System (Amersham). Hybridisation conditions and stringency washesare as described in the AlkPhos Direct kit. Hybridized probe is detectedby chemiluminescence. Hybridisation intensities for each cDNA fragmenton both blots are measured by film densitometry or direct measurement(BioRad Fluor-S Max). The ratio of the tumour to normal hybridisationintensities (T/N) is calculated for each gene to evaluate the degree ofover-expression in the tumour. Genes which are significantlyover-expressed in colon tumours are followed-up. Significance isarbitrarily defined as one standard deviation of the T/N frequencydistribution. Differential screening experiments are repeated using RNAfrom multiple patient donors (>18) to estimate the frequency ofover-expressing tumours in the patient population.

In addition, the DNA arrays are hybridised with mixed cDNA probes fromnormal tissues other than colon (see list above) to determine the levelof expression of the candidate gene in these tissues.

Example 3

DNA Microarrays

DNA micro-arrays are used to examine mRNA expression profiles of largecollections of genes in multiple samples. This information is used tocomplement the data obtained by real-time PCR and provides anindependent measure of gene expression levels in tumors and normaltissues.

Examples of current technologies for production of DNA micro-arraysinclude 1) The Affymetrix “GeneChip” arrays in which oligonucleotidesare synthetized on the surface of the chip by solid phase chemicalsynthesis using a photolithographic process 2) DNA spotting technologyin which small volumes of a DNA solution are robotically deposited andthen immobilized onto the surface of a solid phase (e.g. glass). In bothinstances, the chips are hybridized with cDNA or cRNA which has beenextracted from the tissue of interest (e.g. normal tissue, tumour etc .. . ) and labeled with radioactivity or with a fluorescent reportermolecule. The labeled material is hybridized to the chip and the amountof probe bound to each sequence on the chip is determined using aspecialized scanner. The experiment can be set-up with a singlefluorescent reporter (or radioactivity) or, alternatively, can beperformed using two fluorescent reporters. In this latter case, each ofthe two samples is labeled with one of the reporter molecules. The twolabeled samples are then hybridized competitively to the sequences onthe DNA chip. The ratio of the two fluorescent signals is determined foreach sequence on the chip. This ratio is used to calculate the relativeabundance of the transcript in the two samples. Detailed protocols areavailable from a number of sources including “DNA Microarrays: Apractical approach. Schena M. Oxford University Press 1999” and theWorld Wide Web (http://cmgm.stanford.edu/pbrown/protocols/index.html),http://arrayit.com/DNA-Microarray-Protocols/) and specializeddistributors (e.g. Affymetrix).

Example 5

Northern-Southern Blot Analysis

Limited amounts of mixed tumour and matched normal colon cDNA areamplified by Advantage PCR (see above). Messenger RNA from multiplenormal tissues is also amplified using the same procedure. The amplifiedcDNA (1 μg) is electrophoresed on a 1.2% agarose gel and transferredonto a nylon membrane. The membrane is hybridised (AlkPhos DirectSystem) with a probe prepared using a fragment of the candidate TAAcDNA. Northern-Southern analysis provides information on transcriptsize, presence of splice variants and transcript abundance in tumour andnormal tissues.

Example 6

Northern Blot Analysis

Northern blots are produced according to standard protocols using 1 μgof poly A+ mRNA. Radioactive probes are prepared using the Ready-to-Gosystem (Pharmacia).

Example 7

Experimental Identification of the Full Length cDNA Sequence

Colon tumour cDNA libraries are constructed using the Lambda Zap IIsystem (Stratagene) from 5 μg of polyA+ mRNA. The supplied protocol isfollowed except that SuperscriptII (Life Technologies) is used for thereverse transcription step. Oligo dT-primed and random-primed librariesare constructed. About 1.5×10⁶ independent phages are plated for eachscreening of the library. Phage plaques are transferred onto nylonfilters and hybridised using a cDNA probe labelled with AlkPhos Direct.Positive phages are detected by chemiluminescence. Positive phage areexcised from the agar plat, eluted in 500 μl SM buffer and confirmed bygene-specific PCR. Eluted phages are converted to single strand M13bacteriophage by in vivo excision. The bacteriophage is then convertedto double strand plasmid DNA by infection of E. coli. Infected bacteriaare plated and submitted to a second round of screening with the cDNAprobe. Plasmid DNA is purified from positive bacterial clones andsequenced on both strands.

When the full length gene cannot be obtained directly from the cDNAlibrary, missing sequence is isolated using RACE technology (MarathonKit, ClonTech.). This approach relies on reverse transcribing mRNA intodouble strand cDNA, ligating linkers onto the ends of the cDNA andamplifying the desired extremity of the cDNA using a gene-specificprimer and one of the linker oligonucleotides. Marathon PCR products arecloned into a plasmid (pCRII-TOPO, InVitrogen) and sequenced.

The polynucleotide of SEQ ID NO:1 was obtained using this procedure.

Example 8

EST Profiles

A complementary approach to experimental antigen tissue expressioncharacterization is to explore the human EST database. ESTs (‘ExpressedSequence Tags) are small fragments of cDNA made from a collection ofmRNA extracted from a particular tissue or cell line. Such databasecurrently provides a massive amount of human ESTs (2 10⁶) from severalthousands of cDNA tissue libraries, including tumoral tissues fromvarious types and states of disease. By means of informatics tools(Blast), a comparison search of the CASB7439 sequence is performed inorder to have further insight into tissue expression.

EST Distribution of CASB7439:

EST GenBank Accession number EST cDNA tissue library C00634 Human adult(K. Okubo) AA468668 NCI_CGAP_Co3 AA565752 NCI_CGAP_Co11 AA565766NCI_CGAP_Co11 AA565767 NCI_CGAP_Co11 AI337239 NCI_CGAP_Co16 AI337448NCI_CGAP_Co16 AI393930 NCI_CGAP_CLL1 AI473673 NCI_CGAP_Co14 AI632444NCI_CGAP_GC6 AI861937 NCI_CGAP_Co16 AI825214 NCI_CGAP_GC6 AW080652NCI_CGAP_Co19 AW083899 NCI_CGAP_Co19 AW206058 NCI_CGAP_Sub3 AW237006NCI_CGAP_GC6 AW364626 DT0036 AW449612 NCI_CGAP_Sub5

These ESTs match perfectly with CASB7439. The list contains 9 ESTs from4 different tumor colon libraries, one EST from one normal colonlibrary, 3 ESTs from one tumor germ cell library, one EST from onechronic lymphocyte leukemia cells library, 2 ESTs from 2 mixed tumorslibraries, 2 ESTs from libraries of unknown type. This clearly suggests,as expected, that CASB7439 is over-expressed in tumor tissues, with anemphasis in colorectal tumor tissues, as compared to normal tissues.

Example 9

9.1 Expression and Purification of Tumour-Specific Antigens

Expression in microbial hosts, or alternatively in vitrotranscription/translation, is used to produce the antigen of theinvention for vaccine purposes and to produce protein fragments or wholeprotein for rapid purification and generation of antibodies needed forcharacterization of the naturally expressed protein byimmunohistochemistry or for follow-up of purification.

Recombinant proteins may be expressed in two microbial hosts, E. coliand in yeast (such as Saccharomyces cerevisiae or Pichia pastoris). Thisallows the selection of the expression system with the best features forthis particular antigen production. In general, the recombinant antigenwill be expressed in E. coli and the reagent protein expressed in yeast.

The expression strategy first involves the design of the primarystructure of the recombinant antigen. In general an expression fusionpartner (EFP) is placed at the N terminal extremity to improve levels ofexpression that could also include a region useful for modulating theimmunogenic properties of the antigen, an immune fusion partner (IFP).In addition, an affinity fusion partner (AFP) useful for facilitatingfurther purification is included at the C-terminal end.

As mentioned above, several constructs might undergo comparativeevaluation:

For rapid expression and purification as well as generation ofantibodies against CASB7439, it is proposed to generate in E. Coli afull length CASB7439 protein with NS1 as EFP and a histidine tail asAFP.

Therefore, two constructs are proposed:

Construct 1: Full length wild type CASB7439 cDNA in fusion with NS1 cDNAas EFP and with a histidine tail coding cDNA as an AFP (SEQ ID NO:8).The encoded fusion protein sequence is SEQ ID NO:10.

Construct 2: Full length mutated CASB7439 cDNA in fusion with NS1 cDNAas EFP and with a histidine tail coding cDNA as an AFP (SEQ ID NO:9). Itis proposed in this construct to have the first 50 codons of nativeCASB7439 cDNA replaced by codons specific of the E. coli codon usage, toenhance expression potential of CASB7439 in its E. coli host. Theencoded fusion protein sequence is SEQ ID NO:10.

The CASB7439 protein design is as shown below:

“NS1” is the N-terminal fragment (80 mino acids) of the Influenzaprotein NS1. “HIS” is a polyhistidine tail.

The recombinant strain used is AR58: a cryptic λ lysogen derived fromN99 that is gal E::Tn 10,Δ-8(chlD-pgl),Δ-H1(cro-chlA),N⁺, and cI857(Proc. Natl. Acad. Sci. USA vol 82, pp. 88-92, January 1985Biochemistry)

When the recombinant strains are available, the recombinant product ischaracterized by the evaluation of the level of expression and theprediction of further solubility of the protein by analysis of thebehavior in the crude extract.

After growth on appropriate culture medium and induction of therecombinant protein expression, total extracts are analyzed by SDS-PAGE.The recombinant proteins are visualized in stained gels and identifiedby Western blot analysis using specific antibodies.

Plasmid:

name: TCM 281 pRIT . . . 15143 replicon: pMB1 selection: Kan promotor: PL long insert: NS1-C74-39-His

Expression of the recombinant protein from construct 1:

-   -   Bacteria was grown in LB medium+50 μg/ml Kan at 30° C.

When the culture reached OD=0.5 (620 nm), the culture was heated up to39° C., after 5hours of induction, cells were harvested

Extract Preparation:

Cell concentration: .50X . . . in buffer PBS + complete . . .Disruption: press french 3X Centrifugation: 30 min at 14000 tComment: >90% in the supernatant of cellular extract

The cell extract was run on a 12.5% SDS PAGE, and subsequently stainedwith Coomassie blue. A Western blot was also performed using ancommercial monoclonal antibody against the poly-histidine tail(Quiagen). The resulting gels (FIGS. 3 and 4), show that the protein isexpressed, and visible in the cell extract supernatant.

The purification scheme follows a classical approach based on thepresence of an His affinity tail in the recombinant protein. In atypical experiment the disrupted cells are filtered and the acellularextracts loaded onto an Ion Metal Affinity Chromatography (IMAC; Ni⁺⁻NTAfrom Qiagen) that will specifically retain the recombinant protein. Theretained proteins are eluted by 0-500 mM imidazole gradient (possibly inpresence of a detergent) in a phosphate buffer.

The supernatant from the harvested culture was denatured in 6M urea,100mM NaH₂PO₄, 10 mM Tris, PH 8, and loaded on a chromatographic columnIMAC Qiagen NTA Ni⁺⁺ under the following conditions:

Equilibration buffer: NaH2Po4 100 mM PH 8 Tris 10 mM Urea 6M Sample:supernatant in urea 6M, 100 mM NaH2Po4, 10 mM Tris Wash buffers: 1)NaH2PO4 100 mM PH 8 Tris 10 mM urea 6 M Imidazole 25 mM 2) NaH2Po4 100mM PH 8 Tris 10 mM Urea 6 mM Imidazole 50 mM Elution buffer: NaH2PO4 100mM PH 5.5 Tris 10 mM Urea 6M Imidazole 500 mM

The eluted protein in 500 mM imidazole+6M urea is dialysed under thefollowing conditions:

-   -   PBS PH 7,2+sarkosyl 0.5%+4M urea    -   idem at 2M urea 2 hrs    -   idem at 0M urea 2 hrs

The final material is freezed and stored. The protein content wasquantified using a Lowry protein assay (0.9 mg/1.2 ml). The purity wasassessed by a 12.5% PAGE SDS stained with Coomassie blue (FIG. 5), andthe presence of the recombinant protein was checked by Western blot,using a anti-polyhistidine monoclonal antibody (FIG. 6)

A comparative evaluation of the different versions of the expressedantigen will allow the selection of the most promising candidate that isto be used for further purification and immunological evaluation.

9.2 Antibody Production and Immunohistochemistry

Small amounts of relatively purified protein can be used to generateimmunological tools in order to

a) detect the expression by immunohistochemistry in normal or cancertissue sections;

b) detect the expression, and to follow the protein during thepurification process (ELISA/Western Blot); or

c) characterise/quantify the purified protein (ELISA).

9.2.1 Polyclonal Antibodies:

Immunization

Rabbits are immunised, intramuscularly (I.M.), 3 times at 3 weeksintervals with 100 μg of protein, formulated in the adjuvant3D-MPL/QS21. Three weeks after each immunisation a blood sample is takenand the antibody titer estimated in the serum by ELISA using the proteinas coating antigen following a standard protocol.

ELISA

96 well microplates (maxisorb Nunc) are coated with 5 μg of proteinovernight at 4° C. After 1 hour saturation at 37° C. with PBS NCS 1%,serial dilution of the rabbit sera is added for 1 H 30 at 37° C.(starting at 1/10). After 3 washings in PBS Tween, anti rabbitbiotinylated anti serum (Amersham)) is added ( 1/5000). Plates arewashed and peroxydase coupled streptavidin ( 1/5000) is added for 30 minat 37° C. After washing, 50 μl TMB (BioRad) is added for 7 min and thereaction then stopped with H2SO4 0.2M. The OD can be measured at 450 nmand midpoint dilutions calculated by SoftmaxPro.

9.2.2 Monoclonal Antibodies:

Immunization

5 BALB/c mice are immunized 3 times at 3 week intervals with 5 μg ofpurified protein. Bleedings are performed 14 days post II and 1 weekpost 3. The sera are tested by Elisa on purified protein used as coatedantigen. Based on these results (midpoint dilution>10000) one mouse isselected for fusion.

Fusion/HATselection

Spleen cells are fused with the SP2/0 myeloma according to a standardprotocol using PEG 40% and DMSO 5%. Cells are then seeded in 96 wellplates 2.5×104-105 cells/well and resistant clones will be selected inHAT medium. The supernatant of these hybridomas will be tested for theircontent in specific antibodies and when positive, will be submitted to 2cycles of limited dilution. After 2 rounds of screening, 3 hybridomaswill be chosen for ascitis production.

9.2.3 Immunohistochemistry

When antibodies are available, immuno staining is performed on normal orcancer tissue sections, in order to determine:

-   -   the level of expression of the antigen of the invention in        cancer relative to normal tissue or    -   the proportion of cancer of a certain type expressing the        antigen    -   if other cancer types also express the antigen    -   the proportion of cells expressing the antigen in a cancer        tissue

Tissue Sample Preparation

After dissection, the tissue sample is mounted on a cork disk in OCTcompound and rapidly frozen in isopentane previously super cooled inliquid nitrogen (−160° C.). The block will then be conserved at −70° C.until use. 7-10 μm sections will be realised in a cryostat chamber (−20,−30° C.).

Staining

Tissue sections are dried for 5 min at room Temperature (RT), fixed inacetone for 10 min at RT, dried again, and saturated with PBS 0.5% BSA5% serum. After 30 min at RT either a direct or indirect staining isperformed using antigen specific antibodies. A direct staining leads toa better specificity but a less intense staining whilst an indirectstaining leads to a more intense but less specific staining.

9.3 Analysis of Human Cellular Immune Responses to the Antigen of theInvention

The immunological relevance of the antigen of the invention can beassessed by in vitro priming of human T cells. All T cell lymphocytelines and dendritic cells are derived from PBMCs (peripheral bloodmononuclear cells) of healthy donors (preferred HLA-A2 subtype). AnHLA-A2.1/Kb transgenic mouse model is also used for screening ofHLA-A2.1 peptides.

Newly discovered antigen-specific CD8 T cell lines are raised andmaintained by weekly in vitro stimulation. The lytic activity and theγ-IFN production of the CD8⁺ lines in response to the antigen or antigenderived-peptides is tested using standard procedures.

Two strategies to raise the CD8⁺ T cell lines are used: a peptide-basedapproach and a whole gene-based approach. Both approaches require thefull-length cDNA of the newly discovered antigen in the correct readingframe to be either cloned in an appropriate delivery system or to beused to predict the sequence of HLA binding peptides.

Peptide-Based Approach

Briefly, transgenic mice are immunized with adjuvanted HLA-A2 peptides,those unable to induce a CD8⁺ response (as defined by an efficient lysisof peptide-pulsed autologous spleen cells) will be further analyzed inthe human system.

Human dendritic cells (cultured according to Romani et al.) will bepulsed with peptides and used to stimulate CD8⁺-sorted T cells (byFacs). After several weekly stimulations, the CD8⁺ lines will be firsttested on peptide-pulsed autologous BLCL (EBV-B transformed cell lines).To verify the proper in vivo processing of the peptide, the CD8⁻ lineswill be tested on cDNA-transfected tumour cells (HLA-A2 transfectedLnCaP, Skov3 or CAMA tumour cells).

Whole Gene-Based Approach

CD8⁺ T cell lines will be primed and stimulated with either gene-guntransfected dendritic cells, retrovirally transduced B7.1-transfectedfibroblasts, recombinant pox virus or adenovirus infected dendriticcells. Virus infected cells are very efficient to present antigenicpeptides since the antigen is expressed at high level but can only beused once to avoid the over-growth of viral T cells lines.

After alternated stimulations, the CD8⁻ lines are tested oncDNA-transfected tumour cells as indicated above. Peptide specificityand identity is determined to confirm the immunological validation.

CD4⁺ T-Cell Response

Similarly, the CD4⁺ T-cell immune response can also be assessed.Generation of specific CD4⁺ T-cells is made using dendritic cells loadedwith recombinant purified protein or peptides to stimulate the T-cells.

Predicted Epitopes (Nonamers and Decamers) Binding HLA Alleles:

The HLA Class I binding peptide sequences are predicted either by theParker's algorithm (Parker, K. C., M. A. Bednarek, and J. E. Coligan.1994. Scheme for ranking potential HLA-A2 binding peptides based onindependent binding of individual peptide side-chains. J. Immunol.152:163 and http://bimas.dcrt.nih.gov/molbio/hla_bind/) or the Rammenseemethod (Rammensee, Friede, Stevanovic, MHC ligands and peptide motifs:1st listing, Immunogenetics 41, 178-228, 1995; Rammensee, Bachmann,Stevanovic: MHC ligands and peptide motifs. Landes Bioscience 1997, andhttp://134.2.96.221/scripts/hlaserver.dll/home.htm). Peptides are thenscreened in the HLA-A2.1/Kb transgenic mice model (Vitiello et al.).

The HLA Class II binding peptide sequences are predicted using theTepitope algorithm, with a score cut-off set to 6 (Sturniolo, Hammer atal., Nature Biotechnology. 1999. 17; 555-561).

The following tables gather the Class I and II predicted epitopesequences:

HLA-A 0201: decamers Start Subsequence Parker's Rank PositionResidue Listing Score^(o) SEQ ID: 1 64 KLVNLGFQAL 142.060 SEQ ID NO: 16^(o): Estimate of Half Time of Disassociation of a Molecule ContainingThis Subsequence.

HLA-A 0201: nonamers Start Subsequence Parker's Rank PositionResidue Listing Score^(o) SEQ ID: 1 182 ELLDFSSWL 507.976 SEQ ID NO: 172 104 RLLAEHDAV 126.098 SEQ ID NO: 18 3  64 KLVNLGFQA 100.850SEQ ID NO: 19 ^(o): Estimate of Half Time of Disassociation of aMolecule Containing This Subsequence.

HLA-A 24: nonamers Start Subsequence Parker's Rank PositionResidue Listing Score^(o) SEQ ID: 1 97 EYIRALQRL 360.000 SEQ ID NO: 20^(o): Estimate of Half Time of Disassociation of a Molecule ContainingThis Subsequence.

HLA-A 24: decamers Start Subsequence Parker's Rank PositionResidue Listing Score^(o) SEQ ID: 1 97 EYIRALQLL 360.000 SEQ ID NO: 21^(o): Estimate of Half Time of Disassociation of a Molecule ContainingThis Subsequence.

HLA-B 7: decamers Start Subsequence Parker's Rank PositionResidue Listing Score^(o) SEQ ID: 1 111 AVRNALAGGL 600.000 SEQ ID NO: 22^(o): Estimate of Half Time of Disassociation of a Molecule ContainingThis Subsequence.

HLA-B 4403: decamers Start Subsequence Parker's Rank PositionResidue Listing Score^(o) SEQ ID: 1 156 SEPGSPRSAY 360.000 SEQ ID NO: 232  89 VETLRSAVEY 180.000 SEQ ID NO: 24 ^(o): Estimate of Half Time ofDisassociation of a Molecule Containing This Subsequence.

HLA-DRB1*1501: nonamers Start Subsequence Tepitope Rank PositionResidue Listing Score SEQ ID: 1 99 IRALQRLLA 5.6 SEQ ID NO: 25

HLA-DRB1*1502: nonamers Start Subsequence Tepitope Rank PositionResidue Listing Score SEQ ID: 1 99 IRALQRLLA 4.6 SEQ ID NO: 25

HLA-DRB1*0402: nonamers Start Subsequence Tepitope Rank PositionResidue Listing Score SEQ ID: 1 120 LRPQAVRPS 5.4 SEQ ID NO: 26

HLA-DRB1*1101: nonamers Start Subsequence Tepitope Rank PositionResidue Listing Score SEQ ID: 1 99 IRALQRLLA 4.8 SEQ ID NO: 25

HLA-DRB1*1102: nonamers Start Subsequence Tepitope Rank PositionResidue Listing Score SEQ ID: 1 120 LRPQAVRPS 6.2 SEQ ID NO: 26

HLA-DRB1*1104: nonamers Start Subsequence Tepitope Rank PositionResidue Listing Score SEQ ID: 1 99 IRALQRLLA 5.8 SEQ ID NO: 25

HLA-DRB1*1106: nonamers Start Subsequence Tepitope Rank PositionResiude Listing Score SEQ ID: 1 99 IRALQRLLA 5.8 SEQ ID NO: 25

HLA-DRB1*1301: nonamers Start Subsequence Tepitope Rank PositionResidue Listing Score SEQ ID: 1 120 LRPQAVRPS 6.6 SEQ ID NO: 26 2 73LRQHVPHGG 4.9 SEQ ID NO: 27 3 31 LLRCSRRRR 4.4 SEQ ID NO: 33

HLA-DRB1*1302: nonamers Start Subsequence Tepitope Rank PositionResidue Listing Score SEQ ID: 1 120 LRPQAVRPS 5.6 SEQ ID NO: 26

HLA-DRB1*1304: nonamers Start Subsequence Tepitope Rank PositionResidue Listing Score SEQ ID: 1 120 LRPQAVRPS 6.2 SEQ ID NO: 26 2 73LRQHVPHGG 4.8 SEQ ID NO: 27 3 31 LGFQALRQH 4.6 SEQ ID NO: 28

HLA-DRB1*1305: nonamers Start Subsequence Tepitope Rank PositionResidue Listing Score SEQ ID: 1 99 IRALQRLLA 4.8 SEQ ID NO: 25

HLA-DRB1*0703: nonamers Start Subsequence Tepitope Rank PositionResidue Listing Score SEQ ID: 1 112 VRNALAGGL 5.1 SEQ ID NO: 29 2 98YIRALQRLL 4.8 SEQ ID NO: 30 3 65 LVNLGFQAL 4.5 SEQ ID NO: 31

HLA-DRB5*0101: nonamers Start Subsequence  Tepitope Rank PositionResidue Listing Score SEQ ID: 1 96 VEYIRALQR 4.3 SEQ ID NO: 32

Example 10

CASB7439 Specific Cellular Immune Response

A further way of assessing CASB7439 immunogenicity is to demonstrateCASB7439 antigen has a potential to trigger a cellular immune response.For that purpose, it has to be verified the human CD4+T-cell repertoirehas the ability to recognise CASB7439 antigens presented by APC's(antigen-presenting cells) in a MHC class II restricted manner.

To demonstrate CASB7439 antigen can generate a specific CD4+T-cellactivity in human, as well as to identify CASB7439 epitopes, a series of“in vitro priming” experiments have been carried out with the PBMC's ofthree healthy donors.

In vitro Priming of Donor #1

In-vitro priming cultures with the PBMC of donor #1 were establishedusing 15-mer peptides overlapping by 11 amino acids from the sequence ofCASB7439.

CD4 Peptide Table Peptide Amino SEQ No. Sequence Acids Pool ID NO.  1MDGGTLPRSAPPAPP  1-15 1 34  2 TLPRSAPPAPPVPVG  5-19 1 35  3SAPPAPPVPVGCAAR  9-23 1 36  4 APPVPVGCAARRRPA 13-27 1 37  5PVGCAARRRPASPEL 17-31 1 38  6 AARRRPASPELLRCS  21-35 1 39  7RPASPELLRCSRRRR 25-39 1 40  8 PELLRCSRRRRPATA 29-43 2 41  9RCSRRRRPATAETGG 33-47 2 42 10 RRRPATAETGGGAAA 37-51 2 43 11ATAETGGGAAAVARR 41-55 2 44 12 TGGGAAAVARRNERE 45-59 2 45 13AAAVARRNERERNRV  49-63 2 46 14 ARRNERERNRVKLVN 53-67 2 47 15ERERNRVKLVNLGFQ 57-71 3 48 16 NRVKLVNLGFQALRQ 61-75 3 49 17LVNLGFQALRQHVPH 65-79 3 50 18 GFQALRQHVPHGGAS 69-83 3 51 19LRQHVPHGGASKKLS 73-87 3 52 20 VPHGGASKKLSKVET 77-91 3 53 21GASKKLSKVETLRSA 81-95 3 54 22 KLSKVETLRSAVEYI 85-89 4 55 23VETLRSAVEYIRALQ  89-103 4 56 24 RSAVEYIRALQRLLA  93-107 4 57 25EYIRALQRLLAEHDA  97-111 4 58 26 ALQRLLAEHDAVRNA 101-115 4 59 27LLAEHDAVRNALAGG 105-119 4 60 28 HDAVRNALAGGLRPQ 109-123 5 61 29RNALAGGLRPQAVRP 113-127 5 62 30 AGGLRPQAVRPSAPR 117-131 5 63 31RPQAVRPSAPRGPPG 121-135 5 64 32 VRPSAPRGPPGTTPV 125-139 5 65 33APRGPPGTTPVAASP 129-143 5 66 34 PPGTTPVAASPSRAS 133-147 6 67 35TPVAASPSRASSSPG 137-151 6 68 36 ASPSRASSSPGRGGS 141-155 6 69 37RASSSPGRGGSSEPG 145-159 6 70 38 SPGRGGSSEPGSPRS 149-163 6 71 39GGSSEPGSPRSAYSS 153-167 6 72 40 EPGSPRSAYSSDDSG 157-171 7 73 41PRSAYSSDDSGCEGA 161-175 7 74 42 YSSDDSFCEGALSPA 165-179 7 75 43DSGCEGALSPAEREL 169-183 7 76 44 EGALSPAERELLDFS 173-187 7 77 45SPAERELLDFSSWLGGY 177-193 7 78

Peptides were combined in pools of 6 or 7 peptides/pool (the resulting45 peptide sequences and 7 pools are detailed in table 5), and pulsedonto autologous dendritic cells (DCs). Following 4 stimulation cycles,donor #1 PBMC cell lines were assayed for proliferation by using a3H-thymidine incorporation assay and for IFN-γ synthesis by ELISA.

A number of positive peptide pools were identified, with 21 and 7 cellslines exhibited stimulation index (S.I.)>3 and S.I.>5, respectively(Stimulation index reflects the ratio of activity from T cells incubatedwith DC pulsed with relevant vs. irrelevant peptide or protein).

All positive 21 lines were further re-stimulated with individualpeptides. One line, designated 3H8, showed a specific reactivity topeptide #21 (SEQ ID NO:54).

To map the particular epitope within peptide 21 (SEQ ID NO:54) andrecognised by the T-cells of donor #1, T-Cell clones ere individualizedfrom the T-cell lines. For this purpose, the line 3H8 was re-stimulatedon antigen expanded using polyclonal activator PHA, and cloned on PHA.Clones were then tested for peptide stimulation in IFN-y ELSA assays.

Several clones from line 3H8 were shown to recognize peptide 21 (SEQ IDNO:54). Clones generated from this 3H8 line recognised peptide butfailed to recognise E. coli-derived NS1-CASB7439 protein. Therefore asimilar in vitro priming procedure with a new donor has been undertakento generate T-clones able to recognise the whole CASB7439 proteinpresented by APC's.

In vitro Priming of Donor #2

In vitro priming experiments were preformed with the PBMC from anadditional donor #2 in similar experimental condition as donor #1. Inbrief, PBMC were stimulated with autologous DC pulsed with the 7 poolsof 6-7 peptides at a concentration of 250 ng/ml for each peptide. 29cells lines showed reactivity to pooled peptides, and were furtherassayed on individual peptides (at 250 ng/ml) and on E. coli-derivedNS1-CASB7439 protein (10 μg/ml).

5 of these lines (lines 3A3, 4C5, 3C9, 4D5, and 4B12) demonstratedspecific reactivity to a particular peptide pool and to both anindividual peptide derived from that pool and E. coli-derivedNS1-CASB7439.

Two of the lines, 3A3 and 4C5, have been cloned using PHA. The resultingclones were assayed against DCs pulsed with E. coli-derived NS1-CASB7439fusion protein (2.5 μg/ml) or with irrelevant protein (OspA: 2.5 μg/ml)and assayed for proliferation (3H-Thy) as well as IFN-γ production. 61clones from the two cell lines were shown to recognise E. coli-derivedprotein, and all of the 61 clones have been also shown to recognise thepeptide 16 (SEQ ID NO: 49) from the pool.

These 16 clones were further characterised by demonstrating MHC Class IIrestriction. CD4+clones derived from line 3A3 were assayed against DCspulsed with E. coli-derived NS1-CASB7439 protein or irrelevant protein(OspA: 1 μg/ml) in the presence or absence of antibodies (25 μg/ml) toClass I (W632), Class II (HB145), HLA-DR (L243), or HLA-DQw3 (HB144).These T cell clones were shown to be restricted by MHC Class II, andmore precisely are not DR DQw3 restricted. Moreover, a preliminary donormismatch analyses suggest that these clones are likely restricted byHLA-DQ0602.

Specificity of the NS1-CASB7439 fusion protein generated CD4+ T cellactivity is further demonstrated: indeed, a line 3A3 clone CD4 responsetiters out with CASB7439 protein. No response to OspA, an irrelevantprotein used as negative control, is observed.

In vitro Priming of Donor #3

In vitro priming cultures were established from an additional donorusing the same pools of 15-mer peptide overlapping 11 amino acids andthe same procedures. Four CD4+lines, including 4A7 and 4E4, demonstratedE. coli-derived recombinant protein reactivity that was blocked byantibody to MHC Class II.

Furthermore, CD4+ clones derived from lines 4A7 and 4E4 were assayedagainst dendritic cells pulsed with E. coli-derived NS1-CASB7439 proteinor irrelevant protein (OspA: 10 μg/ml), or CASB7439 peptides (250 ng/ml)in the presence or absence of antibodies to Class I (W632:25 μg/ml) oranti-HLA-DR (L243:25 μg/ml). CD4+ clones derived from these linespresent a specificity that is different from the clones described above,as they are HLA-DR restricted. Moreover, 3 other peptides were shown tobe recognised by CD4+T cells (peptides 23 (SEQ ID NO:56), 24 (SEQ IDNO:57), and 25 (SEQ ID NO:58)), the total number recognised peptide thatwere identified being 5 so far.

Example 11

Immunohistochemical Analysis of CASB7439 on Tumour and Normal ColonBiopsies.

CASB7439 protein over-expression in colon tumour was verified byimmunohistochemistry (IHC) using a CASB7439 specific rabbit polyclonalantibody (Ab CASB7439 #599, 1/50 dilution) directed against an affinitypurified CASB7439 α-peptide (SB599 α-peptide, amino acids 1 to 14 ofCASB7439).

Ab CASB7439 #599 was generated as follows: a rabbit is immunised withSB599 synthetic α-peptide that is conjugated to a carrier protein (LKH).Conjugate is formulated with Freund's adjuvant, and two rabbits areimmunised with formulated conjugate. Four weeks after the secondimmunisation and four weeks after the third immunisation, blood samplesare taken. Anti-CASB7439 antibody titers are estimated in the serum byELISA.

For IHC, paraffin-embedded formalin fixed tissue was sliced into 8micron sections. Steam heat induced epitope retrieval (SHIER) in 0.1 Msodium citrate buffer (pH 6.0) was used for optimal staining conditions.Sections were incubated with 10% serum/PBS for 5 minutes. Tenmicrograms/ml of primary antibody (SB599) was added to each section for25 min followed by a 25 min incubation with a biotinylated anti-rabbitantibody. Endogenous peroxidase activity was blocked by three 1.5 minincubations with hydrogen peroxidase. The avidin biotin complex/horseradish peroxidase (ABV/HRP) system was used along with DAB chromogen tovisualise antigen expression. Slides were counterstained withhematoxylin.

FIGS. 7 and 8 shows IHC results on colon tumour #9476 biospy and colonnormal mucosa #9476, respectively. Anti-CASB7439 immunoreactivity wasobserved at high level in colon cancer and in normal colon at very lowlevel. Anti-CASB7439 immunoreactivity was localised to the cytoplasm andassociated with the plasma membrane of the cells.

Example 12

Demonstration of CASB7439 in vivo Immunogenicity.

Besides being highly tumour specific, the second critical criterion fora candidate vaccine evaluation is its immunogenicity. One way ofassessing the immunogenicity of a protein is to immunise naïve animalswith synthetic peptides derived from the antigen sequence, and whichreproduce natural epitopes. The generated anti-peptide antibodies willthen tend to recognise the native antigen, therefore, showing a specificimmune response can be raised against the candidate vaccine antigen.

Because of the very nature of the immune response, a classical doserange analysis is replaced by repeated immunisations, allowingcomparison of antibody titers before immunisation (control), and aftercumulative injections.

Two peptides were selected from CASB7439 antigen sequence for theirimmunogenic potential: peptides spanning from amino-acids 1-14(peptide 1) and amino-acids 157-172 (peptide 2). Two rabbits wereimmunised with each peptide, rabbits SB598 and SB599 with peptide 1 andrabbits SB600 and SB601 with peptide 2.

The selected peptides were conjugated to a carrier protein (KLH).Rabbits were intramuscularly immunised with CASB7439 peptide, 3 times at3 to 4 weeks intervals with 200 μg of conjugate formulated with Freund'sadjuvant. Four weeks after the second immunisation (PP) and four weeksafter the third immunisation (GP), blood samples were taken, and theCASB7439 specific antibody titers were estimated in the serum by ELISA.Thus, a dose range of 0, 200 and 400 μg of the antigen conjugate isreproduced.

ELISAs were done in triplicate for each immunising peptide and rabbitserum, and performed as follows: 96 well microplates were coated at 4°C. during 16 hours with either 100 ng of CASB7439 peptide or 100 ng ofKLH as coating antigens. After 2-hour saturation at 25° C. with BSA (1mg/ml), serial dilution of the rabbit sera was added for 2 hours at 25°C. (starting at dilution 1/100). Anti rabbit antibody, conjugated withuniversal-HRP, was then added at dilution 1/1000 for 2 hours at 25° C.as secondary antibody. Plates were washed and OPD (0.4 mg/ml) is addedfor 30 min at 25° C. Reaction was stopped with H2SO44M, and OD measuredat 492 nm.

ELISA results clearly show an antibody immune response against distinctCASB7439 synthetic peptide can be raised in several rabbits in adose-dependant manner. This suggests CASB7439 candidate antigen isindeed immunogenic and, when properly formulated with adjuvant as avaccine, CASB7439 vaccine is able to induce a strong and specificantibody immune response.

Sequence Information

SEQ ID NO: 1 GTACCTTGCTTTGGGGGCGCACTAAGTACCTGCCGGGAGCAGGGGGCGCACCGGGAACTCGCAGATTTCGCCAGTTGGGCGCACTGGGGATCTGTGGACTGCGTCCGGGGGATGGGCTAGGGGGACATGCGCACGCTTTGGGCCTTACAGAATGTGATCGCGCGAGGGGGAGGGCGAAGCGTGGCGGGAGGGCGAGGCGAAGGAAGGAGGGCGTGAGAAAGGCGACGGCGGCGGCGCGGAGGAGGGTTATCTATACATTTAAAAACCAGCCGCCTGCGCCGCGCCTGCGGAGACCTGGGAGAGTCCGGCCGCACGCGCGGGACACGAGCGTCCCACGCTCCCTGGCGCGTACGGCCTGCCACCACTAGGCCTCCTATCCCCGGGCTCCAGACGACCTAGGACGCGTGCCCTGGGGAGTTGCCTGGCGGCGCCGTGCCAGAAGCCCCCTTGGGGCGCCACAGTTTTCCCCGTCGCCTCCGGTTCCTCTGCCTGCACCTTCCTGCGGCGCGCCGGGACCTGGAGCGGGCGGGTGGATGCAGGCGCGatggacggcggcacactgcccaggtccgcgccccctgcgccccccgtccctgtcggctgcgctgcccggcggagacccgcgtccccggaactgttgcgctgcagccggcggcggcgaccggccaccgcagagaccggaggcggcgcagcggccgtagcgcggcgcaatgagcgcgagcgcaaccgcgtgaagctggtgaacttgggcttccaggcgctgcggcagcacgtgccgcacggcggcgccagcaagaagctgagcaaggtggagacgctgcgctcagccgtggagtacatccgcgcgctgcagcgcctgctggccgagcacgacgccgtgcgcaacgcgctggcgggagggctgaggccgcaggccgtgcggccgtctgcgccccgcgggccgccagggaccaccccggtcgccgcctcgccctcccgcgcttcttcgtccccgggccgcgggggcagctcggagcccggctccccgcgttccgcctactcgtcggacgacagcggctgcgaaggcgcgctgagtcctgcggagcgcgagctactcgacttctccagctggttagggggctactgaGCGCCCTCGACCTATGAGCCTCAGCCCCGGAAGCCGAGCGAGCGGCCGGCGCGCTCATCGCCGGGGAGCCCGCCAGGTGGACCGGCCCGCGCTCCGCCCCCAGCGAGCCGGGGACCCACCCACCACCCCCCGCACCGCCGACGCCGCCTCGTTCGTCCGGCCCAGCCTGACCAATGCCGCGGTGGAAACGGGCTTGGAGCTGGCCCCATAAGGGCTGGCGGCTTCCTCCGACGCCGCCCCTCCCCACAGCTTCTCGACTGCAGTGGGGCGGGGGGCACCAACACTTGGAGATTTTTCCGGAGGGGAGAGGATTTTCTAAGGGCACAGAGAATCCATTTTCTACACATTAACTTGAGCTGCTGGAGGGACACTGCTGGCAAACGGAGACCTATTTTTGTACAAAGAACCCTTGACCTGGGGCGTAATAAAGATGACCTGGACCCCTGCCCCCACTATCTGGAGTTTTCCATGCTGGCCAAGATCTGGACACGAGCAGTCCCTGAGGGGCGGGGTCCCTGGCGTGAGGCCCCCGTGACAGCCCACCCTGGGGTGGGTTTGTGGGCACTGCTGCTCTGCTAGGGAGAAGCCTGTGTGGGGCACACCTCTTCAAGGGAGCGTGAACTTTATAAATAAATCAGTTCTGTTTAAAAAAAAAAAAAA AAAAA SEQ ID NO: 2MDGGTLPRSAPPAPPVPVGCAARRRPASPELLRCSRRRRPATAETGGGAAAVARRNERERNRVKLVNLGFQALRQHVPHGGASKKLSKVETLRSAVEYIRALQRLLAEHDAVRNALAGGLRPQAVRPSAPRGPPGTTPVAASPSRASSSPGRGGSSEPGSPRSAYSSDDSGCEGALSPAERELLDFSS WLGGY SEQ ID NO: 3MSAPAARSASGAEAHRSRALSSPLTSWRSRVARAPQDSARLRSRCRPTSRRNAGSRAPSCPRGPGTKKRGRARRRPGWSLAARGAQTAARPAASALPPARCARRRARPAGAAARGCTPRLSAASPPCSASCWRRRAARAAAAPGSPSSPASRGCARAHCAALRPLRRLRSLRWPVAAAGCSATVPGTRVSAGQRSRQGRGAQGARTWAVCRRPSRLHPPARSRSRRAAGRCRQRNRRRRGKLWRPKGASGTAPPGNSPGHAS SEQ ID NO: 4GTACCTTGCTTTGGGGGCGCACTAAGTACCTGCCGGGAGCAGGGGGCGCACCGGGAACTCGCAGATTTCGCCAGTTGGGCGCACTGGGGATCTGTGGACTGCGTCCGGGGGATGGGCTAGGGGGACATGCGCACGCTTTGGGCCTTACAGAATGTGATCGCGCCGAGGGGGAGGGCCGAAGCGTGGCGGGAGGGCGAGGCGAAGGAAGGAGGGCGTGAGAAAGGCGACGGCGGCGGCGCGGAGGAGGGTTATCTATACATTTAAAAACCAGCCGCCTGCGCCGCGCCTGCGGAGACCTGGGAGAGTCCGGCCGCACGCGCGGGACACGAGCGTCCCACGCTCCCTGGCGCGTACGGCCTGCCACCACTAGGCCTCCTATCCCCGGGCTCCAGACGACCTAGGACGCGTGCCCTGGGGAGTTGCCTGGCGGCGCCGTGCCAGAAGCCCCCTTGGGGCGCCACAGTTTTCCCCGTCGCCTCCGGTTCCTCTGCCTGCACCTTCCTGCGGCGCGCCGGGACCTGGAGCGGGCGGGTGGATGCAGGCGCGatggacggcggcacactgcccaggtccgcgccccctgcgccccccgtccctgtcggctgcgctgcccggcggagacccgcgtccccggaactgttgcgctgcagccggcggcggcgaccggccaccgcagagaccggaggcggcgcagcggccgtagcgcggcgcaatgagcgcgagcgcaaccgcgtgaagctggtgaacttgggcttccaggcgctgcggcagcacgtgccgcacggcggcgccagcaagaagctgagcaaggtggagacgctgcgctcagccgtggagtacatccgcgcgctgcagcgcctgctggccgagcacgacgccgtgcgcaacgcgctggcgggagggctgaggccgcaggccgtgcggccgtctgcgccccgcgggccgccagggaccaccccggtcgccgcctcgccctcccgcgcttcttcgtccccgggccgcgggggcagctcggagcccggctccccgcgttccgcctactcgtcggacgacagcggctgcgaaggcgcgctgagtcctgcggagcgcgagctactcgacttctccagctggttagggggctactgaGCGCCCTCGACCTAATAAGCCTCAAGCCCCGGAAACCCGAGCGAACGGGCCGGCGCGCTTCATCGCCGGGGAAGCCCGCCAAGGTGGACCGGGCCCGCGCTCCGCCCCCAGCGAGCCGGGGACCCACCCACCACCCCCCGCACCGCCGACGCCGCCTCGTTCGTCCGGCCCAGCCTGACCAATGCCGCGGTGGAAACGGGCTTGGAGCTGGCCCCATAAGGGCTGGCGGCTTCCTCCGACGCCGCCCCTCCCCACAGCTTCTCGACTGCAGTGGGGCGGGGGGCACCAACACTTGGAGATTTTTCCGGAGGGGAGAGGATTTTCTAAGGGCACAGAGAATCCATTTTCTACACATTAACTTGAGCTGCTGGAGGGACACTGCTGGCAAACGGAGACCTATTTTTGTACAAAGAACCCTTGACCTGGGGCGTAATAAAGATGACCTGGACCCCTGCCCCCACTATCTGGAGTTTTCCATGCTGGCCAAGATCTGGACACGAGCAGTCCCTGAGGGGCGGGGTCCCTGGCGTGAGGCCCCCGTGACAGCCCACCCTGGGGTGGGTTTGTGGGCACTGCTGCTCTGCTAGGGAGAAGCCTGTGTGGGGCACACCTCTTCAAGGGAGCGTGAACTTTATAAATAAATCAGTTCTGTTTAAAAAAAAAAAAAAAAAAAAAACCGAGGGGGGGCCCGGAGCC AACAAA SEQ ID NO: 5GGTAAACAGAACTGATTTATTTATAAAGTTCACGCTCCCTTGAAGAGGTGTGCCCCACACAGGCTTCTCCCTAGCAGAGCAGCAGTGCCCACAAACCCACCCCAGGGTGGGCTGTCACGGGGGCCTCACGCCAGGGACCCCGCCCCTCAGGGACTGCTCGTGTCCAGATCTTGGCCAGCATGGAAAACTCCAGATAGTGGGGGCAGGGGTCCAGGTCATCTTTATTACGCCCCAGGTCAAGGGTTCTTTGTACAAAAATAGGTCTCCGTTTGCCAGCAGTGTCCCTCCAGCAGCTCAAGTTAATGTGTAGAAAATGGATTCTCTGTGCCCTTAGAAAATCCTCTCCCCTCCGGAAAAATCTCCAAGTGTTGGTGCCCCCCGCCCCACTGCAGTCGAGAAGCTGTGGGGAGGGGCGGCGTCGGAGGAAGCCGCAGCCCATTATGGGGCCAGCTCCAAGCCCGTTTCCACCGCGGCATTGGTCAGGCTGGGCGGACGAACGAGGCGGCGTCGGCGGTGCGGGGGGTGGTGGGTGGGTCCCCGGCTCGCTGGGGGCGGAGCAGCGGGCCGGTCCACCTG GCGGGCTCCCCSEQ ID NO: 6 TTTTTTTTTTTTTTTTTTTAAACAGAACTGATTTATTTATAAAGTTCACGCTCCCTTGAAGAGGTGTGCCCCACACAGGCTTCTCCCTAGCAGAGCAGCAGTGCCCACAAACCCACCCCAGGGTGGGCTGTCACGGGGGCCTCACGCCAGGGACCCCGCCCCTCAGGGACTGCTCGTGTCCAGATCTTGGCCAGCATGGAAAACTCCAGATAGTGGGGGCAGGGGTCCAGGTCATCTTTATTACGCCCCAGGTCAAGGGTTCTTTGTACAAAAATAGGTCTCCGTTTGCCAGCAGTGTCCCTCCAGCAGCTCAAGTTAATGTGTAGAAAATGGATTCTCTGTGCCCTTAGAAAATCCTCTCCCCTCCGGAAAAATCTCCAAGTGTTGGTGCCCCCCGCCCCACTGCAGTCGAGAAGCTGTGGGGAGGGGCGGCGTCGGAGGAAGCCGCCAGCCCTTATGGGGCCAGCTCCAAGCCCGTTTCCACCGCGGCATTGGTCAGGCTGGGCCGGACGAACGAGGCGGCGTCGGCGGTGCGGGGGGTGGTGGGTGGGTCCCCGGCTCGCTGGGGGCGGAGCGCGGGCCGGTCCACCTGGCGGGCTCCCCGGCGATGAGCGCGCCGGCCGCTCGCTCGGCTTCCGGGGCTGAGGCTCATAGGTCGAGGGCGCTCAGTAGCCCCCTAACCAGCTGGAGAAGTCGAGTAGCTCGCGCTCCGCAGGACTCAGCGCGCCTTCGCAGCCGCTGTCGTCCGACGAGTAGGCGGAACGCGGGGAGCCGGGCTCCGAGCTGCCCCCGCGGCCCGGGGACGAAGAAGCGCGGGAGGGCGAGGCGGCGACCGGGGTGGTCCCTGGCGGCCCGCGGGGCGCAGACGGCCGCACGGCCTGCGGCCTCAGCCCTCCCGCCAGCGCGTTGCGCACGGCGTCGTGCTCGGCCAGCAGGCGCTGCAGCGCGCGGATGTACTCCACGGCTGAGCGCAGCGTCTCCACCTTGCTCAGCTTCTTGCTGGCGCCGCCGTGCGGCACGTGCTGCCGCAGCGCCTGGAAGCCCAAGTTCACCAGCTTCACGCGGTTGCGCTCGCGCTCATTGCGCCGCGCTACGGCCGCTGCGCCGCCTCCGGTCTCTGCGGTGGCCGGTCGCCGCCGCCGGCTGCAGCGCAACAGTTCCGGGGACGCGGGTCTCCGCCGGGCAGCGCAGCCGACAGGGACGGGGGGCGCAGGGGGCGCGGACCTGGGCAGTGTGCCGCCGTCCATCGCGCCTGCATCCACCCGCCCGCTCCAGGTCCCGGCGCGCCGCAGGAAGGTGCAGGCAGAGGAACCGGAGGCGACGGGGAAAACTGTGGCGCCCCAAGGGGGCTTCTGGCACGGCGCCGCCAGGCAACTCCCCAGGGCACGCGTCCTAGGTCGTCTGGAGCCCGGGGATAGGAGGCCTAGTGGTGGCAGGCCGTACGCGCCAGGGAGCGTGGGACGCTCGTGTCCCGCGCGTGCGGCCGGACTCTCCCAGGTCTCCGCAGGCGCGGCGCAGGCGGCTGGTTTTTAAATGTATAGATAACCCTCCTCCGCGCCGCCGCCGTCGCCTTTCTCACGCCCTCCTTCCTTCGCCTCGCCCTCCCGCCACGCTTCGCCCTCCCCCTCGCGCGATCACATTCTGTAAGGCCCAAAGCGTGCGCATGTCCCCCTAGCCCATCCCCCGGACGCAGTCCACAGATCCCCAGTGCGCCCAACTGGCGAAATCTGCGAGTTCCCGGTGCGCCCCCTGCTCCCGGCAGGTACTTAGTGCGCC CCCAAAGCAAGGTACSEQ ID NO: 7 MCRKWILCALRKSSPLRKNLQVLVPPAPLQSRSCGEGRRRRKPPALMGPAPSPFPPRHWSGWAGRTRRRRRCGGWWVGPRLAGGGARARSTLAGFPGDEARRPVRSGFRGLRLIRSRALSSPLTSWRSRVARAPQDSARLRSRCRPTSRRNAGSRAPSCPRGPGTKKRGRARRRPGWSLAARGAQTAARPAASALPPARCARRRARPAGAAARGCTPRLSAASPPCSASCWRRRAARAAAAPGSPSSPASRGCARAHCAALRPLRRLRSLRWPVAAAGCSATVPGTRVSAGQRSRQGRGAQGARTWAVCRRPSRLHPPARSRSRRAAGRCRQRNRRRRGKLWRPKGASGTAPPGNSPGHAS SEQ ID NO: 8ATGGATCCAAACACTGTGTCAAGCTTTCAGGTAGATTGCTTTCTTTGGCATGTCCGCAAACGAGTTGCAGACCAAGAACTAGGTGATGCCCCATTCCTTGATCGGCTTCGCCGAGATCAGAAATCCCTAAGAGGAAGGGGCAGCACcCTcGGTCTGGACATCGAGACAGCCACACGTGCTGGAAAGCAGATAGtGGAGCGGAttctGAAAGAAGAATCCGATGAGGCACTTAAAATGACCATGGACGGCGGCACACTGCCCAGGTCCGCGCCCCCTGCGCCCCCCGTCCCTGTCGGCTGCGCTGCCCGGCGGAGACCCGCGTCCCCGGAACTGTTGCGCTGCAGCCGGCGGCGGCGACCGGCCACCGCAGAGACCGGAGGCGGCGCAGCGGCCGTAGCGCGGCGCAATGAGCGCGAGCGCAACCGCGTGAAGCTGGTGAACTTGGGCTTCCAGGCGCTGCGGCAGCACGTGCCGCACGGCGGCGCCAGCAAGAAGCTGAGCAAAGTGGAGACGCTGCGCTCAGCCGTGGAGTACATCCGCGCGCTGCAGCGCCTGCTGGCCGAGCACGACGCCGTGCGCAACGCGCTGGCGGGAGGGCTGAGGCCGCAGGCCGTGCGGCCGTCTGCGCCCCGCGGGCCGCCAGGGACCACCCCGGTCGCCGCCTCGCCCTCCCGCGCTTCTTCGTCCCCGGGCCGCGGGGGCAGCTCGGAGCCCGGCTCCCCGCGTTCCGCCTACTCGTCGGACGACAGCGGCTGCGAAGGCGCGCTGAGTCCTGCGGAGCGCGAGCTACTCGACTTCTCCAGCTGGTTAGGGGGCTACactagtggccaccatcaccatcaccattaa SEQ ID NO: 9ATGGATCCAAACACTGTGTCAAGCTTTCAGGTAGATTGCTTTCTTTGGCATGTCCGCAAACGAGTTGCAGACCAAGAACTAGGTGATGCCCCATTCCTTGATCGGCTTCGCCGAGATCAGAAATCCCTAAGAGGAAGGGGCAGCACCCTCGGTCTGGACATCGAGACAGCCACACGTGCTGGAAAGCAGATAGTGGAGCGGATTCTGAAAGAAGAATCCGATGAGGCACTTAAAATGACCATGGACGGCGGCACCCTGCCGCGTTCCGCGCCGCCGGCGCCGCCAGTTCCGGTTGGCTGCGCTGCCCGTCGCCGTCCCGCGTCCCCGGAACTGCTGCGCTGCAGCCGTCGCCGTCGCCCGGCCACCGCAGAGACCGGAGGCGGCGCAGCGGCCGTAGCGCGGCGCAATGAGCGCGAGCGCAACCGCGTGAAGCTGGTGAACTTGGGCTTCCAGGCGCTGCGGCAGCACGTGCCGCACGGCGGCGCCAGCAAGAAGCTGAGCAAGGTGGAGACGCTGCGCTCAGCCGTGGAGTACATCCGCGCGCTGCAGCGCCTGCTGGCCGAGCACGACGCCGTGCGCAACGCGCTGGCGGGAGGGCTGAGGCCGCAGGCCGTGCGGCCGTCTGCGCCCCGCGGGCCGCCAGGGACCACCCCGGTCGCCGCCTCGCCCTCCCGCGCTTCTTCGTCCCCGGGCCGCGGGGGCAGCTCGGAGCCCGGCTCCCCGCGTTCCGCCTACTCGTCGGACGACAGCGGCTGCGAAGGCGCGCTGAGTCCTGCGGAGCGCGAGCTACTCGACTTCTCCAGCTGGTTAGGGGGCTACACTAGTGGCCACCATCACCATCACCATTAA SEQ ID NO: 10MDPNTVSSFQVDCFLWHVRKRVADQELGDAPFLDRLRRDQKSLRGRGSTLGLDIETATRAGKQIVERILKEESDEALKMTMDGGTLPRSAPPAPPVPVGCAARRRPASPELLRCSRRRRPATAETGGGAAAVARRNERERNRVKLVNLGFQALRQHVPHGGASKKLSKVETLRSAVEYIRALQRLLAEHDAVRNALAGGLRPQAVRPSAPRGPPGTTPVAASPSRASSSPGRGGSSEPGSPRSAYSSDDSGCEGALSPAERELLDFSSWLGGYTHGHHHHHH SEQ ID NO: 11MYSTAERSVSTLLSFLLAPPCGTCCRSAWKPKFTSFTRLRSRSLRRATAAAPPPVSAVAGRRRRLQRNSSGDAGLRRAAQPTGTGGAGGADLGSVPPSIAPASTRPLQVPARRRKVQAEEPEATGKTVAPQGGFWHGAARQLPRARVLGRLEPGDRRPSGGRPYAPGSVGRSCPARAAGLSQVSAGAAQAAG F SEQ ID NO: 12MEAHLDWYGVPGLQEASDACPRESCSSALPEAREGANVHFPPHPVPREHFSCAAPELVAGAQGLNASLMDGGALPRLMPTSSGVAGACAARRRQASPELLRCSRRRRSGATEASSSSAAVARRNERERNRVKLVNLGFQALRQHVPHGGANKKLSKVETLRSAVEYIRALQRLLAEHDAVRAALAGGLLTPATPPSDECAQPSASPASASLSCASTSPSPDRLGCSEPTSPRSAYSSEES SCEGELSPMEQELLDFSSWLGGYSEQ ID NO: 13 GCCCGGAGCATGGAAGCACGTCAGCTAGGCCATGAACTGCACCCGGGAGGGGTGGGGGTGGAAGCGCACGGTGTCAGCTTTGCAGAATGTGTACACCAAGGGGAGGGCGAGGCGAAGGAAGGAGGGCGTAAGAAAGGAGGCGGTGGCGGGGCGGAGGAGATTATCTATACTTTTTAAAAAAAAGGAGCCTCTTAGCCGCGTAAAGGAGACTTGGGGAGCGCCTGACAGCACGCGCGGGACACGAGAGTACCACGCTTCCCTACTCTTTTCAGACCTTGACTGGTACGGGGTCCCAGGACTGCAGGAGGCCAGCGACGCGTGCCCTAGGGAGTCCTGCAGCAGTGCCCTGCCTGAGGCCCGTGAAGGTGCAAACGTCCACTTCCCACCGCACCCGGTTCCTCGCGAGCACTTTTCCTGTGCCGCACCAGAACTCGTAGCAGGGGCCCAGGGGCTGAATGCAAGCTTGATGGACGGCGGCGCGCTGCCCAGACTCATGCCCACCTCGTCTGGAGTCGCTGGAGCCTGCGCTGCTCGGCGGAGACAAGCGTCTCCGGAATTGCTGCGCTGCAGCCGGCGGCGGCGATCTGGAGCAACCGAGGCCAGCAGCAGCTCGGCGTCCGTGGCACGCCGCAATGAGCGCGAGCGCAACCGCGTAAAGCTGGTAAACTTGGGCTTCCAGGCGCTGCGGCAGCACGTGCCGCACGGCGGCGCCAACAAGAAGCTGAGTAAGGTGGAGACGCTGCGCTCCGCGGTAGAGTACATTCGTGCGCTGCAGCGGCTGCTCGCAGAGCACGACACGGTGCGGCCGGNGCTCGCTGGGGGGCTGTTAACACCCGCTACTCCGCCGTCCGATGAGTGCACGCAGCCCTCTGCCTCCCCTGCCAGCGGGTCTCTGTCCTGCGCCTCTACGTCTCCGTCCCGGACCCTGGGCTGCTCTGAGCCTACCTCCCCGCGCTCCGCCTACTCGTCGGAGGAAAGCAGCTGCGAGGGAGAGCTAAGCCCGATGGAGCAGGAGCTGCTTGACTTTTCCAGTTGGTTAGGGGGCTACTGA SEQ ID NO: 14MESHFNWYGVPRLQKASDACPRESCSSALPEAREGANVHFPPHPVPREHFSCGAPKPVAGAPALNASLMDGGALPRLVPTSSGVAGACTARRRPPSPELLRCSRRRRSGATEASSSSAAVARRNERERNRVKLVNLGFQALRQHVPNGGANKKLSKVETLRSAVEYIRALQRLLAEHDAVRAALSGGLLTPATRPSDVCTQPSASPASASLSCTSTSPDRLGCSEPASPRSAYSSEDSSC EGETYPMGQMFDFSNWLGGYSEQ ID NO: 15 TTCACCCGGCTGCAAGCGCTAGGTGTACGGAGACCTGGCAGCTCTTGGGGCTTAAGGACTGAGCRCCAGAGCCGGTGGAGGTTCCTGTGGAGTACATTCGGACCCTCTCACAGCCCCCGAGAGTGCGGGACGTGCGGAGCGCAGTTCGGGATCTGCACTCGAGGACTTGTCGAGGACGCATTAAGCTAAGCATCTGCTCGGAGCATGGAATCGCACTTTAACTGGTACGGGGTCCCAAGGCTCCAGAAGGCTAGCGACGCGGTGCCCTAGGGAATCCTGCGCAGTGCCCTGCCTGAGGCCCGTGAAGGTGCGAACGTCCACTTCCCACCGCACCCGGTTCCTCGCGAGCACTTTTCCTGTGGCGCACCGAAACCCGTAGCGGGGGCCCCGGCGCTGAATGCAAGCTTGATGGACGGCGGCGCGCTGCCCAGACTCGTGCCCACCTCGTCTGGAGTCGCTGGAGCCTGCACTGCTCGGCGGAGACCCCCGTCCCCGGAACTGCTTCGCTGCAGCCGACGGCGGCGATCGGGAGCAACCGAGGCCAGCAGCAGCTCGGCGGCCGTGGCACGCCGCAATGAGCGTGAGCGCAACCGCGTAAAGCTGGTAAACTTGGGCTTCCAGGCGCTGCGGCAGCACGTGCCGCACGGCGGCGCCAACAAGAAGCTGAGTAAGGTGGAGACGCTGCGCTCCGCGGTAGAGTACATCCGTGCGCTGCAGCGGCTGCTAGCAGAGCACGACGCGGTGCGTGCTGCGCTCTCTGGGGGTCTATTAACACCCGCTACTCGGCCGTCCGATGTGTGCACGCAGCCCTCCGCCTCCCCTGCCAGCGCGTCTCTGTCCTGCACCTCTACATCCCCAGACCGCCTAGGCTGCTCCGAGCCTGCCTCTCCGCGCTCCGCCTACTCGTCGGAGGACAGCAGCTGCGAGGGAGAGACTTACCCGATGGGGCAGATGTTTGACTTTTCCAATTGGTTAGGGGGCTACTGAGCACCCCACACCCCTAAGCTGCGTCCCTGGGTGTCCCCTGGTGGACCTACCTGCGTTTCTTGCCCAGGAAACCTGGGCCCATGCCTTACCCATGCTGTCTAGTGCAGCCTGACCAAATGCCAAGTACTGACCTCTGCTCGGCCTCCACGCCGCGGAATGACATCTTCCATCTCCCAGTCCTTGCCGAACCAGGACTTGGAAATTTCTCAGGAGAAAGAATTTTACAATGACAATCTGCTTTTTATCAATTAACTTGAACTGCTGGAGGACTCTGCTGAAAATATGAAGAATTATTTTTATACAAAGGATCCTTAAGCTTGGAGCACAATAAAGATGACCTCTGTCTCTCACCCCCACTGTCTAGAACTTTCCAACCTGGCCAAAGTGTGGACGGGTCGGGCCCTGAGGGCAAGATGCCTGGCTGCACCCTTCTTCCTCTTCCGAAGCCTATCCTGACGCTGATGTTTGGCCAGTGTGGGAACCCTGCTATTGCAAAGTGTACTATTCTATAAAAGTTGTTTTTCATTGGAAAGGAATTC SEQ ID NO: 16 KLVNlGFQALSEQ ID NO: 17 ELLDFSSWL SEQ ID NO: 18 RLLAEHDAV SEQ ID NO: 19 KLVNLGFQASEQ ID NO: 20 EYIRALQRL SEQ ID NO: 21 EYIRALQRLL SEQ ID NO: 22AVRNALAGGL SEQ ID NO: 23 SEPGSPRSAY SEQ ID NO: 24 VETLRSAVEYSEQ ID NO: 25 IRALQRLLA SEQ ID NO: 26 LRPQAVRPS SEQ ID NO: 27 LRQHVPHGGSEQ ID NO: 28 LGFQALRQH SEQ ID NO: 29 VRNALAGGL SEQ ID NO: 30 YIRALQRLLSEQ ID NO: 31 LVNLGFQAL SEQ ID NO: 32 VEYIRALQR SEQ ID NO: 33 LLRCSRRRR

1-33. (canceled)
 34. An immunogenic composition comprising one or more isolated polypeptides consisting of a 9-15 amino acid fragment of SEQ ID NO:2, wherein said fragment encompasses an amino acid sequence selected from SEQ ID NOs:16-33, wherein the composition, when administered to a subject, induces an immune response that recognizes a polypeptide having the sequence of SEQ ID NO:2.
 35. The immunogenic composition of claim 34 further comprising an adjuvant.
 36. The immunogenic composition of claim 35 wherein the adjuvant comprises one or more components selected from the group consisting of 3D-MPL, QS21, a mixture of QS21 and cholesterol, and a CpG oligonucleotide.
 37. A method for inducing an immune response in a mammal comprising administration of an immunogenic composition comprising one or more isolated polypeptides consisting of a 9-15 amino acid fragment of SEQ ID NO:2, wherein said fragment encompasses an amino acid sequence selected from SEQ ID NOs:16-33, wherein the composition, when administered to a subject, induces an immune response that recognizes a polypeptide having the sequence of SEQ ID NO:2.
 38. The method of claim 37, wherein the mammal is human. 